Methods and compositions for generating recombinant nucleic acid molecules

ABSTRACT

A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Ser. No. 60/520,946, filed Nov. 17, 2003; and is aContinuation-in-Part of U.S. Ser. No. 10/014,128, filed Dec. 7, 2001;which claims the benefit of priority under 35 U.S.C. §119(e) of U.S.Ser. No. 60/326,092, filed Sep. 28, 2001, and U.S. Ser. No. 60/254,510,filed Dec. 8, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to compositions and methods forfacilitating the construction of recombinant nucleic acid molecules, andmore specifically to compositions for using one or more topoisomerasesto generate covalently closed recombinant nucleic acid molecules and tomethods of making such recombinant nucleic acid molecules.

2. Background Information

The advent of recombinant DNA technology has allowed the cloning andidentification of genes from many different organisms, and thedetermination of the complete genomes of an ever-increasing number oforganisms, including humans. The elucidation of a large number of newand uncharacterized genes creates a pressing need for technologies thatenable rapid expression and analysis of these genes. The ability toconstruct recombinant nucleic acid molecules has provided a means toproduce novel “gene products” and to express gene products, particularlyheterologous gene products, in cells, tissues and organisms in whichthey are not normally produced. Thus, recombinant DNA technology hasled, for example, to the fields of gene therapy, in which defectivegenes are replaced by copies of a normal gene; and “biopharming,” inwhich, for example, a gene product such as an antibody, which normallyis produced by an animal, is expressed in a plant, thereby allowinglarge scale production of the gene product.

Despite the great leaps in progress that have resulted from thediscovery and development of recombinant DNA methods, a great number ofsteps often is required to prepare a novel DNA construct having desiredproperties. A significant bottleneck in recombinant DNA methodology isthe requirement that each nucleic acid sequence that is to be used toprepare a construct must be cloned into a vector, the vector must beintroduced into and amplified in a host cell (generally a bacterialcell), the amplified vector must be isolated from the host cell, andthen must be transformed or transfected into the appropriate cell typefor expression. Vectors with the appropriate functional elements such asa promoter, an origin of replication, a selectable marker, an epitopetag, or the like may need to be constructed. Such methods requiremultiple restriction enzyme digestion and ligation steps, in addition tonumerous purification and characterization steps.

Methods and products are being developed to reduce the number of stepsrequired to obtain a desired nucleic acid construct. For example, manycommercial suppliers provide vectors that contain one or more functionalelements of interest, and have cloning sites such that a desirednucleotide sequence can be cloned in frame with the sequences in thevector. However, such vectors are limited in that only the most commonlyused elements such as particularly useful promoters or tags or the likecan be included in the vectors in order for the vector to becommercially viable.

In some cases, there may be no need to covalently ligate togethernucleic acid sequences that have been allowed to join. For example,non-covalently linked constructs formed by hybridization ofcomplementary overhanging ends can be used to transfect cells with areasonably high efficiency. However, such constructs effectively contain“nicks” at the sites of hybridization and, therefore, are moresusceptible to endonuclease degradation than covalently linkedsequences. Furthermore, constructs containing nicks are not suitable forcertain further manipulations such as amplification by a polymerasechain reaction. Thus, a need exists to identify methods for facilitatingthe preparation of nucleic acid constructs. The present inventionsatisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to methods of covalently linking, in oneor both strands, two or more double stranded (ds) nucleotide sequencesusing one or more topoisomerases. As such, the invention also provides,in part, nucleotide sequences that can be covalently linked according tosuch methods, recombinant nucleic acid molecules generated therefrom,and compositions comprising the nucleotide sequence and/or recombinantnucleic acid molecules (e.g., reaction mixtures), wherein the nucleotidesequences contain at least one topoisomerase attached thereto (e.g., acovalently linked topoisomerase), at least one topoisomerase recognitionsite, or a combination thereof.

In particular embodiments, at least one topoisomerase recognition sitecan be internal, i.e., within one or more nucleotide sequences, or canbe at or near one or both termini of a single stranded nucleotidesequence or one or both strands of double stranded nucleotide sequence;or at least one bound topoisomerase can be at or near one or bothtermini of a single stranded nucleotide sequence or one or both strandsof a double stranded nucleotide sequence, and can be present on 5′overhang, a 3′ overhang, or at a blunt end. For example, one or more ofthe at least one topoisomerase or the at least one topoisomeraserecognition site can be located at or near a 5′ terminus, at or near a3′ terminus, at or near both 5′ termini, at or near both 3′ termini, ator near a 5′ terminus and a 3′ terminus, at or near a 5′ terminus andboth 3′ termini, or at or near a 3′ terminus and both 5′ termini. Theinvention provides methods for preparing and using nucleotide sequencesand covalently linked recombinant nucleic acid molecules generatedtherefrom, compositions containing one or more of such nucleotidesequences or recombinant nucleic acid molecule, and nucleic acidmolecules and compositions derived therefrom. In specific aspects, theinvention provides nucleotide sequences 1) to which topoisomerases ofvarious types (e.g., a type IA topoisomerase, a type IB topoisomerase, atype II topoisomerase, etc.) are attached (e.g., covalently bound);and/or 2) which contain two or more topoisomerase recognition sites thatcan be bound and/or cleaved by various types of topoisomerases; and/or3) which contain a combination of such bound various topoisomerases andvarious topoisomerase recognition sites, as well as methods forpreparing and using compositions comprising such nucleotide sequences.

The invention further provides methods for covalently linking two ormore nucleotide sequences, wherein at least one of the nucleotidesequences contains at least one topoisomerase bound thereto or onetopoisomerase recognition site. Further, when nucleotide sequences usedin methods of the invention contain more than one topoisomerase, eitheron the same or different nucleotide sequences, the topoisomerase can beof the same type or of different types. Similarly, when nucleotidesequences used in methods of the invention contain more than onetopoisomerase recognition site, either on the same or differentnucleotide sequences, the topoisomerase recognition sites can berecognized by topoisomerases of the same type or of different types.Thus, the invention provides methods for covalently linking nucleotidesequences employing any one topoisomerase or topoisomerase recognitionsite. The invention also provides methods for covalently linkingnucleotide sequences using any combination of topoisomerases and/ortopoisomerase recognition sites. The invention also provides covalentlylinked recombinant nucleic acid molecules produced by such methods, andfurther provides compositions containing such recombinant nucleic acidmolecules and uses of these molecules.

The present invention generally provides, in part, methods forcovalently linking any number of nucleotide sequences (e.g., two, three,four, five, six, seven, eight, nine, ten, etc.), including nucleotidesequences containing different functional or structural elements. Assuch, the invention provides, in part, methods for covalently linkingany number of nucleotide sequences (e.g., two, three, four, five, six,seven, eight, nine, ten, etc.) that confer different properties upon acovalently linked recombinant nucleic acid molecule generated therefrom.In many instances, the methods of the invention result in the formationof recombinant nucleic acid molecules having operative interactions ofproperties and/or elements of individual nucleotide sequences that arecovalently linked to generate the recombinant nucleic acid molecules(e.g., an operative interaction/linkage between an expression controlelement and an open reading frame). Examples of 1) functional andstructural elements and 2) properties that can be conferred upon arecombinant nucleic acid molecule generated according to a method of theinvention include, but are not limited to, multiple cloning sites (e.g.,nucleotide sequences that contain at least two restriction endonucleasecleavage sites), packaging signals (e.g., viral packaging signals suchas adenoviral packaging signals, alphaviral packaging signals, etc.),restriction endonuclease cleavage sites, open reading frames (e.g.,intein coding sequences, affinity purification tag coding sequences,etc.), expression control sequences (e.g., promoters, operators, etc.),and the like. Additional elements and properties that can be conferredby one or more nucleotide sequences upon a product recombinant nucleicacid molecule are exemplified herein or otherwise known in the art. Thepresent invention also provides covalently linked recombinant nucleicacid molecules produced by the methods described above, as well as usesof these molecules and compositions containing these molecules.

The invention also includes in vivo and in vitro methods for generatingRNA molecules. In specific embodiments, the invention includes methodsfor the in vitro generation of RNA molecules. In certain aspects, thesemethods may comprise, for example, (a) generating a reaction mixture inwhich a first double-stranded DNA molecule is contacted with a seconddouble-stranded DNA molecule under conditions which allow for bothstrands of one end of the first double-stranded DNA molecule to becomecovalently linked to both strands of one end (i.e., the 5′ and 3′termini) of the second double-stranded DNA molecule, (b) incubating thereaction mixture of (a) for a sufficient period of time to allow for thecovalent linking of the first double-stranded DNA molecule to the seconddouble-stranded DNA molecule, and (c) generating an RNA transcript fromthe product of (b) by in vitro transcription. Also, the firstdouble-stranded DNA molecule may have promoter activity and may beoperably connected to the second double-stranded DNA molecule in (b).Further, the first double-stranded DNA molecule and the seconddouble-stranded DNA molecule may be covalently linked to each other by atopoisomerase. Additionally, the double-stranded DNA molecule producedin (b) may not contain a nick in either strand at the position where thefirst double-stranded DNA molecule and the second double-stranded DNAmolecule are joined. In related methods, both strands of one end (i.e.,the 5′ and 3′ termini) of the double-stranded DNA molecule product of(b) may be covalently linked by a topoisomerase to both strands of oneend (i.e., the 5′ and 3′ termini) of a third double-stranded DNAmolecule. In additional aspects, the double-stranded DNA molecules maybe covalently linked in the order of the first double-stranded DNAmolecule, the second double-stranded DNA molecule, and the thirddouble-stranded DNA molecule. In specific instances, the thirddouble-stranded DNA molecule may encode a polyadenylation signal and/orthe second double-stranded DNA molecule may encode a polypeptide.Further, the second double-stranded DNA molecule may be generated bypolymerase chain reaction. Additionally, one strand of each end of thefirst double-stranded DNA molecule and the second double-stranded DNAmolecule which are joined may be topoisomerase-charged. Further, thetopoisomerase may be a type IB topoisomerase or a catalytic domain of atype IB topoisomerase.

In related methods, transcription products generated by methods of theinvention may be either translated in vitro to generate a polypeptide orintroduced into cells, where they may be translated in vivo.

Further, promoters used in methods of the invention (e.g., methodinvolving in vitro transcription) include T7 and T3 promoters.

When more than one RNA molecule is generated in methods of theinvention, the individual RNA molecules may be generated in separatereaction vessels or may be generated in the same reaction vessel.Further, these individual RNA molecules may share sufficient sequencecomplementarity to allow for them to hybridize to each other. Typically,this will result in the formation of RNA which is at least partiallydouble-stranded. Along these lines, the invention includes methods forthe in vitro generation of double-stranded RNA molecules, these methodmay comprise, for example, (a) generating a reaction mixture in which afirst double-stranded DNA molecule is contacted with a seconddouble-stranded DNA molecule under conditions which allow for bothstrands of one end of the first double-stranded DNA molecule to becomecovalently linked to both strands of a first end of the seconddouble-stranded DNA molecule, (b) incubating the reaction mixture of (a)for a sufficient period of time to allow for the covalent linking of thefirst double-stranded DNA molecule to the first end of the seconddouble-stranded DNA molecule, and (c) generating a reaction mixture inwhich a first double-stranded DNA molecule is contacted with a seconddouble-stranded DNA molecule under conditions which allow for bothstrands of one end of the first double-stranded DNA molecule to becomecovalently linked to both strands of a second end of the seconddouble-stranded DNA molecule, (d) incubating the reaction mixture of (c)for a sufficient period of time to allow for the covalent linking of thefirst double-stranded DNA molecule to the second end of the seconddouble-stranded DNA molecule, (e) mixing the products of (b) and (d),(f) generating RNA transcripts (e.g., sense and antisense RNA molecules)of the products of (e) by in vitro transcription, and (g) incubating theRNA transcripts produced in (f) under conditions which allow for theformation of double-stranded RNA molecules. Further, the firstdouble-stranded DNA molecule may have promoter activity and may beoperably connected to each of the second double-stranded DNA moleculesin (b) and (d). Also, the first double-stranded DNA molecule and thesecond double-stranded DNA molecule may be covalently linked to eachother by a topoisomerase. Additionally, the double-stranded DNA moleculeproduced in (b) may not contain a nick in either strand at the positionwhere the first double-stranded DNA molecule and the seconddouble-stranded DNA molecule are joined. Further, the seconddouble-stranded DNA molecule may encode a polypeptide. Additionally, oneor more of the double-stranded DNA molecules (e.g., the seconddouble-stranded DNA molecule) may be generated by polymerase chainreaction. Also, in methods of the invention, one strand of each of theends of the double stranded DNA molecules which are joined (e.g., thefirst double-stranded DNA molecule and the second double-stranded DNAmolecule) may be topoisomerase-charged. In such instances, thetopoisomerase may be, for example, a type IB topoisomerase or acatalytic domain of a type IB topoisomerase.

The invention further includes reaction mixtures for performing methodsof the invention and/or containing molecules generated by methods of theinvention. The invention thus includes, for example, reaction mixturescomprising (a) a first double-stranded DNA molecule which comprises apromoter, and (b) a second double-stranded DNA molecule. Also, onestrand of one end of the first double-stranded DNA molecule may betopoisomerase-charged. Further, one strand of one end of the seconddouble-stranded DNA molecule may be topoisomerase-charged. Additionally,topoisomerase-charged ends of the first double-stranded DNA molecule andthe second double-stranded DNA molecule may be capable of hybridizing toeach other.

The invention also provides compositions that contain nucleotidesequences and/or recombinant nucleic acid molecules as disclosed herein.For example, compositions of the invention include, but are not limitedto, mixtures (e.g., reaction mixtures) containing a nucleotide sequencecomprising at least one topoisomerase recognition site, and at least onetopoisomerase that recognizes at least one of the at least onetopoisomerase recognition sites of the nucleotide sequence. Compositionsof the invention further include at least one nucleotide sequencecomprising 1) at least one topoisomerase recognition site or at leastone nucleotide sequence to which at least one topoisomerase is attached(e.g., covalently bound) and 2) one or more additional components.Examples of such additional components include, but are not limited to,topoisomerases; additional nucleotide sequence that can, but need not,comprise one or more topoisomerases or topoisomerase recognition sites;buffers; salts; polyamines (e.g., spermine, spermidine, etc.); water; orany other component as disclosed herein or as desired.

In one embodiment, the invention provides a method of using atopoisomerase (e.g., a type IA or type IB topoisomerase) to covalentlylink a first ds nucleotide sequence to at least a second ds nucleotidesequence, thereby generating a recombinant ds nucleic acid molecule thatis covalently linked in at least one strand. Such a method can be used,for example, to covalently link three or more (e.g., 3, 4, 5, 6, 7,etc.) ds nucleotide sequences, so as to generate a recombinant dsnucleic acid molecule containing one strand that has no nicks. Inparticular embodiments of a method of generating a recombinant doublestranded nucleic acid molecule that is covalently linked in only onestrand, the topoisomerase is not a type IB topoisomerase.

In another embodiment, the invention provides a method of using a typeIA topoisomerase and a type IB topoisomerase to covalently link at leasttwo ds nucleotide sequences in at least one strand. For example, a firstds nucleotide sequence can contain a type IA topoisomerase at the 5′terminus of one end and a type IB topoisomerase at the 3′ terminus ofthe second end of the same strand, thereby providing a means tocovalently link a strand of the first ds nucleotide sequence to one ormore other ds nucleotide sequences to generate a recombinant ds nucleicacid molecule that is covalently linked in one strand. In anotherembodiment, the present invention provides a method to covalently linktwo or more ds nucleotide sequences in both strands, for example, bycontacting an end of a first ds nucleotide sequence having a type IA ora type IB topoisomerase bound thereto, to an end of a second dsnucleotide sequence having a type IA or type IB topoisomerase,respectively, bound thereto; or by contacting a first ds nucleotidesequence having a type IA topoisomerase and a type IB topoisomerasebound to the 5′ terminus and 3′ terminus, respectively, of an end, witha second ds nucleotide sequence. The invention also providescompositions comprising nucleic acid molecules with topoisomerase boundto a 5′ terminus and/or a 3′ terminus, as well as precursor nucleotidesequences having one or more topoisomerase recognition sites forpreparing covalently linked recombinant nucleic acid molecules having atopoisomerase bound to a 5′ and/or 3′ terminus.

The present invention also relates to methods of generating a doublestranded recombinant nucleic acid molecule, which is covalently linkedin one or both strands, by contacting two or more (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, etc) ds nucleotide sequences with at least one (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, etc.) topoisomerase. For example, the presentinvention provides methods for generating a ds recombinant nucleic acidmolecule covalently linked in both strands, and methods for generating ads recombinant nucleic acid molecule covalently linked in at least onestrand.

A method for generating a ds recombinant nucleic acid molecule that iscovalently linked in one strand generally is performed by contacting asite-specific topoisomerase (e.g., a type IA or type IB topoisomerase)and at least one (e.g., 1, 2, 3 4, 5, 6, 7, 8, 9, 10, etc.) dsnucleotide sequences to be joined under conditions such that at leastone strand of an end of each ds nucleotide sequence is covalently linkedto at least one strand of an end of any one or two other ds nucleotidesequences. Such a method can be used to generate, for example, a dsrecombinant nucleic acid molecule, wherein one strand contains a nick atthe site or sites at which the substrate ds nucleotide sequences areligated. The present invention also provides recombinant nucleic acidmolecules prepared by such a method, further provides nucleotidesequences used in such a method.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in at least one strand can be performed using variouscombinations of components. For example, the method can be performed bycontacting at least one (e.g., 1, 2, 3 4, 5, 6, 7, 8, 9, 10, etc.)substrate ds nucleotide sequence to be linked and at least one (e.g., 1,2, 3 4, 5, 6, 7, 8, 9, 10, etc.) site-specific topoisomerase (e.g., atype IA topoisomerase or type IB topoisomerase), wherein thetopoisomerase cleaves one or both strands of the substrate ds nucleotidesequences and forms a stable complex with a nucleotide at a terminus ofthe cleavage site. The topoisomerase-charged end ortopoisomerase-charged ds nucleotide sequence is then contacted withanother end or ds nucleotide sequence, which is, or can be, charged witha topoisomerase, (e.g., a type IA or type IB topoisomerase) such thatone strand, but not both strands, at one or both ends of the substrateds nucleotide sequences is linked, thereby generating one or more dsrecombinant nucleic molecules covalently linked in one strand. Thesite-specific type IA topoisomerase, and type IB topoisomerase whenpresent, links one strand of each ds nucleotide sequence through theformation of a phosphodiester bond at each linkage site.

A method of generating a ds recombinant nucleic acid molecule that iscovalently linked in at least one strand also can be performed bycontacting at least one site-specific topoisomerase-charged dsnucleotide sequence (e.g., a ds nucleotide sequence charged with a typeIA or a type IB topoisomerase), with at least a secondtopoisomerase-charged ds nucleotide sequence (e.g., a ds nucleotidesequence charged with a type IA or a type IB topoisomerase); or bycontacting at least one topoisomerase-charged ds nucleotide sequence(e.g., a ds nucleotide sequence charged with a type IA or type IBtopoisomerase) with at least one ds nucleotide sequence that contains atopoisomerase cleavage site, in the presence of excess topoisomerase; orby contacting at least one site-specific topoisomerase-charged dsnucleotide sequence (e.g., a ds nucleotide sequence charged with a typeIA or a type IB topoisomerase) with at least one ds nucleotide sequence;or by contacting at least one ds nucleotide sequence that contains asite-specific topoisomerase cleavage site (e.g., a type IA or type IBtopoisomerase cleavage site), and at least one ds nucleotide sequence,in the presence of an excess of site-specific topoisomerase (e.g., typeIA or type IB topoisomerase, respectively). The present invention alsoprovides recombinant nucleic acid molecules prepared by such a method,as well as compositions for performing such methods. Such compositionsinclude, for example, a topoisomerase-charged ds nucleotide sequence,wherein topoisomerase is covalently linked to one or both 5′ termini; a5′ terminus and one or both 3′ termini; or both 5′ termini and both 3′termini.

Such a method also can be performed by contacting 1) a first dsnucleotide sequence having a first end and a second end, wherein thefirst ds nucleotide sequence has a site-specific topoisomeraserecognition site (e.g., a type IA or type II topoisomerase recognitionsite) at or near the 5′ terminus of the first end or the second end; 2)at least a second ds nucleotide sequence having a first end and a secondend; and 3) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)site specific topoisomerase, under conditions such that all componentsare in contact and the at least one topoisomerase can effect itsactivity. For example, the topoisomerase can be a type IA topoisomerasesuch as E. coli topoisomerase I, E. coli topoisomerase III, or aeukaryotic topoisomerase III. Upon cleavage of the first ds nucleotidesequence, the topoisomerase preferably is stably bound to a 5′ terminus.Preferably, upon cleavage by the topoisomerase, the cleaved dsnucleotide sequence comprises a 3′ overhanging sequence.

The method also can be performed by contacting 1) a first ds nucleotidesequence having a first end and a second end, wherein the first dsnucleotide sequence has a site-specific topoisomerase recognition site(e.g., a type IA or a type II topoisomerase recognition site) at or nearthe 5′ terminus of the first end or the second end or both ends; 2) atleast a second ds nucleotide sequence that has, or can be made to have,a first end and a second end; and 3) at least one (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, etc.) site-specific type IA topoisomerase, underconditions such that all components are in contact and the at least onetopoisomerase can effect its activity. For example, the topoisomerasecan be a type IA topoisomerase such as E. coli topoisomerase I, E. colitopoisomerase III, or a eukaryotic topoisomerase III. Upon cleavage of ads nucleotide sequence, the topoisomerase preferably is stably bound tothe 5′ terminus. Preferably, upon cleavage by the topoisomerase, thecleaved ds nucleotide sequence comprises a 3′ overhanging sequence. Assuch, a method of the invention provides a means wherein any combinationof ends can be linked, and wherein one strand of the product recombinantnucleic acid molecule is covalently linked and the second strand is notcovalently linked (i.e., contains a nick).

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in one strand, involving a first ds nucleotide sequence and atleast a second ds nucleotide sequence, can further include a step ofamplifying the ds recombinant nucleic acid molecule covalently linked inone strand. The amplification can be carried out by contacting the dsrecombinant nucleic acid molecule with an amplification reaction primerpair, wherein a first primer of the primer pair can bind to thecovalently linked strand, at or near one end of the first or second dsnucleotide sequence, and prime an amplification reaction in a directiontoward the other (i.e., second or first, respectively) ds nucleotidesequence to generate a first extension product that is identical innucleotide sequence to the nicked strand of the ds recombinant nucleicacid molecule. The second primer of the primer pair is selected suchthat it can bind to the first extension product, typically at or nearthe 3′ terminus of the first extension product, and, in the presence ofthe first primer, can generate an amplification product using thecovalently-linked strand and the first extension product (or extensionproducts generated therefrom) as templates. For example, the method canbe performed such that the topoisomerase recognition site (e.g., type IAtopoisomerase recognition site) is at or near the first end of the firstds nucleotide sequence, and the method can further include contactingthe generated ds recombinant nucleic acid molecule with an amplificationreaction primer pair, wherein a forward primer is capable of binding ator near the second end of the first ds nucleotide sequence and wherein areverse primer is capable of binding to a nucleotide sequencecomplementary to at least a portion of the second end of the second dsnucleotide sequence; and amplifying the ds recombinant nucleic acidmolecule. By way of example, the first ds nucleotide sequence caninclude a coding region and the second ds nucleotide sequence caninclude a regulatory element, and the generated recombinant nucleic acidmolecule can comprise an expressible nucleotide sequence.

A method for generating a ds recombinant nucleic acid moleculecovalently linked in one strand also can be performed by contacting 1) afirst ds nucleotide sequence having a first end and a second end,wherein the first ds nucleotide sequence has a site-specifictopoisomerase recognition site (e.g., type IA or type II topoisomeraserecognition site) at or near the 5′ terminus of the first end or thesecond end or both; 2) at least a second ds nucleotide sequence thathas, or can be made to have, a first end and a second end; 3) at least athird ds nucleotide sequence which has, or can be made to have, a firstend and a second end, each end further comprising a 5′ terminus and a 3′terminus; and 4) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,etc.) site-specific topoisomerase (e.g., at least one type IAtopoisomerase), under conditions such that all components are in contactand the at least one topoisomerase can effect its activity. For example,the topoisomerase can be a type IA topoisomerase such as E. colitopoisomerase 1, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a ds nucleotide sequence, thetopoisomerase preferably is stably bound to the 5′ terminus. Preferably,upon cleavage by the topoisomerase, the cleaved ds nucleotide sequencecomprises a 3′ overhanging sequence.

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, involving a first dsnucleotide sequence that contains a site-specific topoisomeraserecognition site (e.g., a type IA or type IB topoisomerase recognitionsite), or cleavage product thereof, at least a second ds nucleotidesequence, and at least a third ds nucleotide sequence can be performedsuch that any combination of ends are linked, and one strand at the endsbeing linked is covalently linked and one strand is nicked. Furthermore,in this embodiment, any of the ends can contain a type IA or type IBtopoisomerase recognition site, or cleavage product thereof, providedthat the first ds recombinant nucleotide molecule contains a type IA ortype II topoisomerase recognition site at or near a 5′ terminus, orcleavage product thereof, and only one topoisomerase or topoisomeraserecognition site is present at the ends that are to be linked. Forexample, where the first ds nucleotide sequence comprises a type IAsite-specific topoisomerase recognition site at or near each of saidfirst end and said second end, the method further can include contactingthe first ds nucleotide sequence and the second ds nucleotide sequencewith at least a third ds nucleotide sequence which has, or can be madeto have, a first end and a second end, each end further comprising a 5′terminus and a 3′ terminus, under conditions such that the type IAtopoisomerase can covalently link the 5′ terminus of the first end ofthe first ds nucleotide sequence with the 3′ terminus of the first endof the second nucleotide sequence, and the 5′ terminus of the second endof the first ds nucleotide sequence with the 3′ terminus of the firstend of the third nucleotide sequence. It will be recognized that othercombinations of ends and topoisomerase recognition sites, or cleavageproducts thereof, can be used in practicing a method of the invention.

In another embodiment, a method for generating a ds recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein the first ds nucleotide sequence has a site-specifictopoisomerase recognition site (e.g., a type IA or type II topoisomeraserecognition site) at or near the 5′ terminus of an end and a type IBtopoisomerase recognition site at or near the 3′ terminus of the otherend; 2) at least a second ds nucleotide sequence that has, or can bemade to have, a first end and a second end; 3) at least one (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, etc.) site-specific type IA topoisomerase; and4) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) type IBtopoisomerase under conditions such that all components are in contactand the at least one topoisomerase can effect its activity. For example,the topoisomerase recognition site at or near a 5′ terminus of the firstds nucleotide sequence can be a type IA topoisomerase such as E. colitopoisomerase I, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a ds nucleotide sequence, the typeIA topoisomerase preferably is stably bound to the 5′ terminus, and thetype IB topoisomerase preferably is stably bound at the 3′ terminus.Preferably, upon cleavage by the topoisomerases, the cleaved dsnucleotide sequence comprises a 3′ overhanging sequence and a 5′overhanging sequence.

Methods of the invention can further include contacting the dsrecombinant nucleic acid molecule with one or more (e.g., 1, 2, 3, 4, 5,etc.) enzymes or agents having ligase activity (e.g., a DNA ligase suchas T4 DNA ligase) 1) to covalently link gaps, particularly nicks, in oneor both strands of the product ds recombinant nucleic acid molecule toobtain a ds recombinant nucleic acid molecule covalently linked in bothstrands; 2) to link a product ds nucleic acid molecule to one or moreother molecules; and/or 3) to circularize the product ds recombinantnucleic acid molecule.

A method for generating a ds recombinant nucleic acid moleculecovalently linked in one strand, involving a first ds nucleotidesequence, a second ds nucleotide sequence, and at least a third dsnucleotide sequence, can further include a step for amplifying the dsrecombinant nucleic acid molecule covalently linked in one strand using,for example, an amplification reaction such as a polymerase chainreaction. Such a method can be used to amplify any portion of thegenerated ds recombinant nucleic acid molecule, particularly all or aportion of the covalently linked strand, including a portion of thecovalently linked strand that includes all or a part of each of thesubstrate first, second and third ds nucleotide sequences. For example,where the ds recombinant nucleic acid molecule comprises an end of thefirst ds nucleotide sequence linked to an end of the second dsnucleotide and an end of the third ds nucleotide sequence linked to theother end of the second ds nucleotide sequence, the amplification can becarried out by contacting the ds recombinant nucleic acid molecule withan amplification reaction primer pair, wherein a first primer of theprimer pair is capable of binding to the covalently linked strand at ornear one end of the first or third ds nucleotide sequence and priming anamplification reaction in a direction toward the second ds nucleotidesequence to generate a first extension product that is complementary tothe covalently linked strand; and the second primer of the primer paircan bind to the first extension product, typically at or near the 3′terminus of the first extension product, which can include a sequencecomplementary to at least a portion of the second nucleotide sequenceand can further include a sequence complementary to the third or firstds nucleotide sequence, respectively, and, in the presence of the firstprimer, can generate an amplification product using the covalentlylinked strand and the extension product (or extension products generatedtherefrom) as templates. The method can be performed such that thetopoisomerase recognition site (e.g., type IA or type IB topoisomeraserecognition site) is at or near the first end of the first ds nucleotidesequence, and the method further includes contacting the ds recombinantnucleic acid molecule with an amplification reaction primer pair,wherein a forward primer is capable of binding to a nucleotide sequenceat or near the second end of the first ds nucleotide sequence andwherein a reverse primer is capable of binding to a nucleotide sequencecomplementary to at least a portion of the third ds nucleotide sequence;and amplifying the ds recombinant nucleic acid molecule. By way ofexample, the first ds nucleotide sequence can include a first regulatoryelement such as a transcriptional promoter and/or an operator (e.g., atetracycline operator), the second ds nucleotide sequence can include acoding region, and the third ds nucleotide sequence can include a secondregulatory element such as a transcriptional termination sequence.Furthermore, ends being linked according to a method of the inventioncan contain complementary overhanging sequences. The present inventionalso provides recombinant nucleic acid molecules or amplificationproducts thereof produced using such a method.

Methods of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, are further exemplified bycontacting I) a first ds nucleotide sequence having a first end and asecond end, wherein, at the first end, second end, or both ends, thefirst ds nucleotide sequence has a topoisomerase covalently bound to a5′ terminus (i.e., a topoisomerase-charged 5′ terminus); and 2) at leasta second nucleotide sequence, under condition such that thetopoisomerases can covalently link one strand, but not both strands, ofone or both ends of the first ds nucleotide sequence with one or bothends of at least the second ds nucleotide sequence. The ds nucleotidesequences can contain a 3′ hydroxyl group at the end of a strand beinglinked to a 5′ terminus by topoisomerase, or a 3′ hydroxyl group can begenerated using a phosphatase. As disclosed herein, such a method can beperformed using only a first ds nucleotide sequence and a second dsnucleotide sequence, or can include a third, fourth, fifth, or more dsnucleotide sequences as desired, wherein each nucleotide sequence is asdefined, including optionally comprising one or twotopoisomerase-charged termini. A second (or other) ds nucleotidesequence independently can have a topoisomerase covalently bound to a 5′terminus of one end or at both ends of the ds nucleotide sequence, and,unless indicated otherwise, the first and second (or other) dsnucleotide sequences can be the same or can be different.

Methods of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, are further exemplified bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein, at the first end, second end, or both ends, thefirst ds nucleotide sequence has a topoisomerase covalently bound to a5′ terminus (i.e., a topoisomerase-charged 5′ terminus); 2) at least asecond nucleotide sequence which may or may not be charged withtopoisomerase; and 3) at least a third nucleotide sequence which may ormay not be charged with topoisomerase, under condition such that thetopoisomerases can covalently link one strand, but not both strands, ofone or both ends of the first ds nucleotide sequence with one or bothends of at least the second ds nucleotide sequence, or one or both endsof at least the third ds nucleotide sequence. The ds nucleotidesequences can contain a 3′ hydroxyl group at the end of a strand beinglinked to a 5′ terminus by topoisomerase, or a 3′ hydroxyl group can begenerated using a phosphatase. The second, third, (or other) dsnucleotide sequence independently can have a topoisomerase covalentlybound to a 5′ terminus of one end or at both ends of the ds nucleotidesequence, and, unless indicated otherwise, the first, second, third (orother) ds nucleotide sequences can be the same or can be different.

In another embodiment, a method for generating a ds recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein a site-specific topoisomerase (e.g., a type IA ortype II topoisomerase) is bound at the 5′ terminus of the first end, thesecond end, or both the first end and the second end; and 2) at least asecond ds nucleotide sequence that has, or can be made to have, a firstend and a second end, under conditions such that all components are incontact and the at least one topoisomerase can effect its activity. Forexample, a type IA topoisomerase such as E. coli topoisomerase I, E.coli topoisomerase III, or a eukaryotic topoisomerase III, can be used.The ds nucleotide sequences can include a 3′ overhanging sequence, a 5′overhanging sequence, or can be blunt ended.

In another embodiment, a method for generating a ds recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein a site-specific topoisomerase (e.g., a type IA ortype II topoisomerase) is bound at the 5′ terminus of the first end, thesecond end, or both the first end and the second end; 2) at least asecond ds nucleotide sequence that has, or can be made to have, a firstend and a second end; wherein a site-specific topoisomerase (e.g., atype IA or type II topoisomerase) can be bound at the 5′ terminus of thefirst end, the second end, or both the first end and the second end; and3) at least a third ds nucleotide sequence that has, or can be made tohave, a first end and a second end, wherein a site-specifictopoisomerase (e.g., a type IA or type II topoisomerase) can be bound atthe 5′ terminus of the first end, the second end, or both the first endand the second end; under conditions such that all components are incontact and the at least one topoisomerase can effect its activity. Forexample, the type IA topoisomerase can be E. coli topoisomerase I, E.coli topoisomerase III, or a eukaryotic topoisomerase III. The dsnucleotide sequences can include 3′ overhanging sequences, 5′overhanging sequences, or can be blunt ended, or can have variouscombinations of such ends, which can facilitate directional linkage.

The present invention also relates to a method of generating a dsrecombinant nucleic acid molecule covalently linked in one strand by 1)amplifying a portion of a first ds nucleotide sequence using a PCRprimer pair, wherein at least one primer of the primer pair encodes asite-specific type IA topoisomerase recognition site, thereby producingan amplified first ds nucleotide sequence having a first end and asecond end, wherein the first end, second end, or both ends have atopoisomerase recognition site at or near the 5′ terminus; and 2)contacting a) the amplified first ds nucleotide sequence; b) at least asecond ds nucleotide sequence having a first end and a second end; andc) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) sitespecific type IA topoisomerase, under conditions such that the at leastone topoisomerase can cleave the first and/or second end of theamplified first ds nucleotide sequence having a type IA topoisomeraserecognition site, and can effect its ligating activity. The PCR primerencoding the topoisomerase recognition site can further comprise anucleotide sequence at its 5′ terminus, i.e., 5′ to the topoisomeraserecognition site, such that, upon cleavage of the amplified first dsnucleotide sequence by a site specific topoisomerase, the ds nucleotidesequence contains a 3′ overhanging sequence, which can be complementaryto a 3′ overhanging sequence of a second (or other) ds nucleotidesequence to which the first ds nucleotide sequence is to be linkedaccording to a method of the invention for generating a ds recombinantnucleic acid molecule covalently linked in one strand. A second primerof the PCR primer pair can include the complement of a type IBtopoisomerase recognition site, thereby producing an amplificationproduct having a first end and a second end, wherein the amplificationproduct comprises a type IA topoisomerase recognition site at or nearthe 5′ terminus of one end and a type IB topoisomerase recognition siteat or near the 3′ terminus of the other end.

The present invention further relates to a ds recombinant nucleic acidmolecule having, or which can be made to have, a first end and a secondend, each end including a 5′ terminus and a 3′ terminus, wherein the dsrecombinant nucleic acid molecule comprises a site-specifictopoisomerase recognition site (e.g., type IA topoisomerase recognitionsite) at or near a 5′ terminus of the first end, the second end, or boththe first end and the second end. The ds recombinant nucleic acidmolecule can further include a type IB topoisomerase recognition site ator near a 3′ terminus of an end that does not include a type IAtopoisomerase recognition site. The ds recombinant nucleic acid moleculecan be a vector, or can be a component of a vector, for example, acomponent that allows for convenient insertion of a regulatory elementor an origin of replication or the like.

The present invention also relates to a topoisomerase-charged dsrecombinant nucleic acid molecule having a first end and a second end,each end having a 5′ terminus and a 3′ terminus, wherein a site-specifictype IA topoisomerase is bound at the 5′ terminus of the first end, thesecond end, or both the first end and the second end. For example, thetopoisomerase-charged nucleic acid ds recombinant nucleic acid moleculecan include a type IA topoisomerase bound at the 5′ termini of each ofthe first and second ends. The topoisomerase-charged nucleic acid dsrecombinant nucleic acid molecule can include, for example, a type IBtopoisomerase bound at a 3′ terminus of an end not bound by a type IAtopoisomerase, or can contain a site-specific topoisomerase recognitionsite at an end not bound by a type IA topoisomerase. Thetopoisomerase-charged nucleic acid ds recombinant nucleic acid moleculecan comprise a vector or a component thereof, or can comprise aregulatory element or coding sequence or any other nucleic acid moleculeof interest.

In one aspect, the methods of the invention allow joining of two or morenucleic acid sequences in a desired orientation and/or order, which, ifdesired, can be further manipulated or used in a variety of assays orprocedures, including a transcription or transfection procedure, whichcan be performed in vitro or in vivo, a translation reaction or otherprotein expression procedure, and the like. In another aspect, (1) threeor more, four or more, five or more, etc., or (2) a population orlibrary of the same or different ds nucleotide sequences can be linkedaccording to a method of the invention. In still another aspect, themethods of the invention can be used to link each end of a singlenucleic acid molecule to form a circular or supercoiled molecule. Inaddition, where two or more nucleic acid sequences have been joined, theends of the resulting ds recombinant nucleic acid molecule can becovalently linked in one or both strands according to a method of theinvention to circularize the molecule.

The nucleotide sequences to be linked can be derived from any source,and can be naturally occurring and chemically or recombinantlysynthesized nucleic acid molecules such as cDNA, genomic DNA, plasmids,vectors, oligonucleotides, and the like. Furthermore, the nucleotidesequences can, but need not, contain one or more functional sequencessuch as gene regulatory elements; origins of replication; splice sites;polyadenylation sites; packaging signals; multiple cloning sites; openreading frames, which can encode, for example, tag sequences, detectableor selectable markers, cell localization domains, or other peptide orpolypeptide, or can encode an antisense nucleic acid molecule, ribozyme,tRNA or other RNA molecule; and the like. As such, a method of theinvention allows any number of nucleotide sequences, which can be thesame or different, to be covalently linked in one or both strands,including, if desired, in a predetermined order or orientation or both.

The ds nucleotide sequences to be linked can be in any form, forexample, linear, circular, or supercoiled, and are characterized, inpart, in that each ds nucleotide sequence to be linked is a substratefor a selected topoisomerase or can be modified to be a substrate. Thetopoisomerase can be any topoisomerase that can covalently link onestrand of a ds nucleotide sequence to one strand of another dsnucleotide sequence, preferably through a phosphodiester bond. Thetopoisomerase can be a site specific topoisomerase or can have relaxedspecificity, and preferably forms a stable complex (e.g., a covalentcomplex) with one strand of the ds nucleotide sequence at or near thesite at which cleavage is effected.

In certain aspects, the present invention provides methods forgenerating a ds recombinant nucleic acid molecule that is covalentlylinked in both strands. Such a method can be performed by contactingtopoisomerase and the ds nucleotide sequences to be joined underconditions such that both strands of an end of one ds nucleotidesequence are ligated to both strands of an end of at least one (e.g., 1,2, 3 4, 5, 6, 7, 8, 9, 10, etc.) other ds nucleotide sequence. As such,a method of the invention generates a ds recombinant nucleic acidmolecule that is covalently linked in both strands and, therefore, doesnot contain a nick in either strand at the site or sites at which thesubstrate ds nucleotide sequences are ligated. The present inventionalso provides recombinant nucleic acid molecules prepared according tosuch a method.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in both strands can be performed using various combinations ofcomponents. For example, the method can be performed by contacting twoor more substrate ds nucleotide sequences to be covalently linked and atleast one topoisomerase, wherein the topoisomerase cleaves one or bothstrands of the ds nucleotide sequences and forms a stable complex with anucleotide at a terminus of the cleavage site. The topoisomerase-chargedends or topoisomerase-charged ds nucleotide sequences are then contactedwith each other such that each strand of the substrate ds nucleotidesequences is linked, thereby generating one of more covalently linked dsrecombinant nucleic molecules. Preferably, the topoisomerase mediatesthe formation of a phosphodiester bond at each linkage site. The methodalso can be performed by contacting two or more topoisomerase-charged dsnucleotide sequences, either alone, or in the presence of excesstopoisomerase, or by contacting one or more topoisomerase-charged dsnucleotide sequences with one or more ds nucleotide sequences thatcontain a topoisomerase cleavage site, and a topoisomerase. The presentinvention also provides recombinant nucleic acid molecules prepared bysuch a method.

In various embodiments, the topoisomerase can have a relatively relaxedspecificity such that it can bind to and cleave a variety of differentnucleotide sequences, or the topoisomerase can be a site-specifictopoisomerase, which binds to and cleaves a specific nucleotidesequence. The topoisomerase also can be a type I topoisomerase, whichcleaves one strand of a ds nucleotide sequence, or can be a type IItopoisomerase, which cleaves both strands of a ds nucleotide sequence.Where the topoisomerase is a type I topoisomerase, cleavage is effectedsuch that a linear ds nucleotide sequence is produced, and istopoisomerase-charged at one or both ends. Preferably, the strand of theds nucleotide sequence that is complementary to the strand containingthe bound topoisomerase forms an overhanging sequence.

An advantage of performing a method of the invention is that theligation reaction performed by a topoisomerase occurs very quickly andover a wide range of temperatures. Another advantage of the methods ofthe invention is that generated ds recombinant nucleic acid moleculesthat are covalently linked in one or both strands can be used directlyin a subsequent procedure, for example, as a substrate for anamplification reaction such as a polymerase chain reaction (PCR), or asa substrate for a transcription or translation or coupledtranscription/translation reaction.

By way of example, a method of the invention for generating a dsrecombinant nucleic acid molecule covalently linked in both strands, canbe performed by contacting 1) a first ds nucleotide sequence having afirst end and a second end, wherein, at the first end or second end orboth, the first ds nucleotide sequence has a topoisomerase recognitionsite at or near the 3′ terminus; 2) at least a second ds nucleotidesequence having a first end and a second end, wherein, at the first endor second end or both, the at least second double stranded nucleotidesequence has a topoisomerase recognition site at or near a 3′ terminus;and 3) at least one site specific topoisomerase (e.g., a type IA and/ora type IB topoisomerase), under conditions such that all components arein contact and the topoisomerase can effect its activity. Preferably,the strand complementary to that containing the topoisomeraserecognition sequence comprises a 5′ hydroxyl group, and more preferably,upon cleavage by the topoisomerase, comprises a 5′ overhanging sequence.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in both strands also can be performed by contacting 1) a dsnucleotide sequence having a first end and a second end, wherein each ofthe first end and second end contains a topoisomerase recognition siteat or near the 3′ terminus, and 2) a site specific topoisomerase, underconditions such that the components are in contact and the topoisomerasecan effect its activity. For example, the topoisomerase can be a type IBtopoisomerase such as a Vaccinia topoisomerase or an S. cerevisiaetopoisomerase. Such a method provides a means to prepare a covalentlyclosed circular or supercoiled ds recombinant nucleic acid molecule.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in both strands also can be performed by contacting 1) a first dsnucleotide sequence having a first end and a second end, wherein thefirst ds nucleotide sequence has a topoisomerase recognition site at ornear the 5′ terminus of the first end or the second end or both; 2) atleast a second ds nucleotide sequence having a first end and a secondend, wherein the at least second double stranded nucleotide sequence hasa topoisomerase recognition site at or near the 5′ terminus of the firstend or the second end or both; and 3) at least one site specifictopoisomerase, under conditions such that all components are in contactand the at least one topoisomerase can effect its activity. For example,the topoisomerase can be a type IA topoisomerase such as E. colitopoisomerase I, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a ds nucleotide sequence, thetopoisomerase preferably is stably bound to the 5′ terminus. The 3′terminus of the end containing the topoisomerase recognition site, orbound topoisomerase, can comprise a 3′ hydroxyl group, or can bemodified to comprise a 3′ hydroxyl group. Preferably, upon cleavage bythe topoisomerase, the cleaved ds nucleotide sequence comprises a 3′overhanging sequence.

The methods of the invention as exemplified herein can be performedusing two or more site specific topoisomerases, wherein the first,second or other ds nucleotide sequence substrates correspondingly have,at or near a 3′ terminus or 5′ terminus of an end, a topoisomeraserecognition site for one of the two or more topoisomerases. The use oftwo or more topoisomerases, and corresponding topoisomerase recognitionsites, can facilitate the joining of the ds nucleotide sequences in apredetermined order, orientation, or combination thereof. Thus, it willbe recognized that, where a method of the invention is exemplified usinga topoisomerase, the method similarly can be performed using two or moretopoisomerases. In some cases, reference is made to the use of at leastone topoisomerase, and, unless indicated otherwise, the methods can beperformed using one, two, three or more topoisomerases, provided thesubstrate ds nucleotide sequences contain the appropriate topoisomeraserecognition sites. Similar considerations are relevant totopoisomerase-charged ds nucleotide sequence substrates, including thatthe topoisomerases can be the same or different.

The present invention provides methods for generating a ds recombinantnucleic acid molecule that is covalently linked in both strands. Such amethod can be performed by contacting 1) a first ds nucleotide sequencehaving a first end and a second end, wherein the first ds nucleotidesequence has a topoisomerase recognition site at or near the 3′ terminusand a topoisomerase recognition site at or near the 5′ terminus of thefirst end or of the second end or of both ends; 2) at least a second dsnucleotide sequence having a first end and a second end; and 3) at leasttwo (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site specifictopoisomerases, under conditions such that all components are in contactand each of the topoisomerases can effect its activity. Upon cleavage ofthe termini of the substrate first ds nucleotides sequence by thetopoisomerases, the 5′ terminus or the 3′ terminus of one or both of thefirst or second ends can comprise an overhanging sequence, or can beblunt ended, or a first end can contain an overhang and the second endcan be blunt ended. Where present, an overhanging sequence of a firstend will generally have sufficient complementarity to an overhangingsequence of a second (or other) end to allow for specific hybridizationof the two ends to each other. Further, when the first and second endsare on different molecules, methods of the invention result in the twomolecules becoming linked and when the first and second ends are on thesame molecule, the methods result in the molecule becoming circularized.

The number of different topoisomerases useful in such an embodiment willdepend, in part, on whether the first ds nucleotide sequence containstopoisomerase recognition sites at only the first end or the second end,or contains topoisomerase recognition sites at both ends, and further,where the ds nucleotide sequence contains topoisomerase recognitionsites on both ends, whether the 3′ recognition sites or the 5′recognition sites are different. In addition, the method can beperformed such that one or more of the at least second ds nucleotidesequences also can contain a topoisomerase recognition site at or nearthe 3′ terminus and/or a topoisomerase recognition site at or near the5′ terminus of the first end or of the second end or of both ends,wherein the topoisomerase recognition sites at or near the 3′ terminusor the 5′ terminus or both of the other ds nucleotide sequence can thesame as or different from the topoisomerase recognition sites in thefirst ds nucleotide sequence. As such, the number of differenttopoisomerases further can depend on the number of different substrateds nucleotide sequences being linked according to a method of theinvention.

An advantage of performing a method of the invention using a sitespecific topoisomerase is that the first ds nucleotide sequence, thesecond ds nucleotide sequence, and one or more additional ds nucleotidesequences can be covalently linked, in one or both strands, in apredetermined directional orientation. An additional advantage is that aproduct comprising nucleotide sequences spanning the linkage site can beselected in vitro by performing an amplification reaction using a firstprimer that selectively hybridize to a sequence downstream of thelinkage site and a second primer complementary to a sequence upstream ofthe linkage site, for example, amplification primers specific for thetermini or sequences near the termini of a ds recombinant nucleic acidmolecule covalently linked in both strands. A ds recombinant nucleicacid molecule, covalently linked in one or both strands, generatedaccording to a method of the invention can be used directly in furtherprocedures such as, for example, for transfecting a cell; as a templatefor performing amplification (e.g., PCR); in an in vitro transcriptionreaction; in a coupled transcription/translation reaction; for linkageto other nucleotide sequences using a restriction endonuclease site,which can be contained in a multiple cloning site; or for chromosomalintegration via homologous recombination. Accordingly, a ds recombinantnucleic acid molecule generated according to a method of the inventioncan be useful, without further manipulation, for various purposes.

In an aspect of the invention, the first ds nucleotide sequences arederived from at least a first population of nucleic acid molecules, forexample, from a cDNA library or a combinatorial library such as acombinatorial library of synthetic oligonucleotides, and the second dsnucleotide sequences are derived from at least a second population of dsnucleotide sequences. According to a method of the invention, linking offirst ds nucleotide sequences with second ds nucleotide sequencesprovides a means to generate combinatorial populations of ds recombinantnucleic acid molecules that are covalently linked in one or bothstrands. In accordance with such a method, one or more target nucleicacid molecules also can be linked with the recombinant nucleic acidmolecules of the population to produce additional populations. Suchpopulations of combinatorial molecules can be further manipulated oranalyzed, for example, by protein expression and screening for fusionproteins having desirable characteristics.

In one embodiment, a method of the invention is performed such that thefirst ds nucleotide sequence comprises an open reading frame, forexample, an isolated cDNA or coding sequence or exon of a gene, and asecond ds nucleotide sequence comprises a regulatory element such as apromoter, which can be operatively covalently linked to the 5′ end ofthe coding sequence such that the coding sequence can be transcribedtherefrom. A second ds nucleotide sequence also can comprise two or moreregulatory elements, for example, a promoter, an internal ribosome entrysite and an ATG initiator methionine codon, in operative linkage witheach other, which can be operatively covalently linked to the 5′ end ofa first ds nucleotide sequence comprising a coding sequence according toa method of the invention. Such a method can further include contactinga third ds nucleotide sequence comprising, for example, apolyadenylation signal and/or a suppressible STOP codon, which can beoperatively covalently linked to the 3′ end of the coding sequence. Sucha method can be useful for generating an expressible nucleic acidmolecule, which can be transcribed, translated, or both as a functionalunit. In addition, or alternatively, a ds nucleotide sequence encoding adetectable marker, for example, an epitope tag, can be operativelylinked to a first or second (or other) ds nucleotide sequence accordingto a method of the invention. The generation of a ds recombinant nucleicacid molecule having a desired directional orientation of the nucleotidesequences in such a construct can be facilitated by includingcomplementary 5′ or 3′ overhanging sequences at the termini of the dsnucleotide sequences to be covalently linked together by thetopoisomerase.

In an embodiment, a method of the invention is performed such that atleast the first ds nucleotide sequence or the at least second dsnucleotide sequence is one of a plurality of nucleotide sequences, forexample, a cDNA library, a combinatorial library of nucleotidesequences, or a variegated population of nucleotide sequences. Inanother embodiment, a method of the invention includes furthercontacting a ds recombinant nucleic acid molecule, covalently linked inone or both strands, with a PCR primer pair, and amplifying all or aportion of the covalently linked ds recombinant nucleic acid molecule.In addition to generating a large amount of product, the amplificationreaction can be selective for constructs comprising a desired covalentlylinked ds recombinant nucleic acid molecule, particularly where the dsnucleotide sequences to be covalently linked comprise complementaryoverhanging sequences. As such, a method of the invention provides an invitro selection means that is suitable for high throughput analysis.

A method for generating a ds recombinant nucleic acid moleculecovalently linked in both strands is exemplified by contacting 1) afirst ds nucleotide sequence having a first end and a second end,wherein, at the first end, second end, or both ends, the first dsnucleotide sequence has a topoisomerase covalently bound to the 3′terminus (“topoisomerase-charged”); and 2) at least a second dsnucleotide sequence, which can, but need not, be charged withtopoisomerase. Preferably, the topoisomerase-charged ds nucleotidesequence or sequences contain a 5′ hydroxyl group at the ends having thebound topoisomerase, although 5′ hydroxy groups also can be generatedusing a phosphatase. The methods of the invention can be performed usingonly a first ds nucleotide sequence and a second ds nucleotide sequence,or can include a third, fourth or more ds nucleotide sequences asdesired, wherein each nucleotide sequence is as defined above. A firstor second (or other) ds nucleotide sequence independently can have atopoisomerase covalently bound to a 3′ terminus of one end or at bothends of the nucleotide sequence, and, unless indicated otherwise, thefirst and second (or other) ds nucleotide sequences can be the same orcan be different.

Methods of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in both strands are further exemplified bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein, at the first end, second end, or both ends, thefirst ds nucleotide sequence has a topoisomerase covalently bound to a5′ terminus (i.e., a topoisomerase-charged 5′ terminus); and 2) at leasta second nucleotide sequence, which can, but need not, be charged withtopoisomerase. The topoisomerase-charged ds nucleotide sequence orsequences can contain a 3′ hydroxyl group at the ends containing thebound topoisomerase, or a 3′ hydroxyl group can be generated using aphosphatase. As disclosed herein, such a method can be performed usingonly a first ds nucleotide sequence and a second ds nucleotide sequence,or can include a third, fourth or more ds nucleotide sequences asdesired, wherein each nucleotide sequence is as defined, includingcomprising at least one topoisomerase-charged 5′ terminus. A first orsecond (or other) ds nucleotide sequence independently can have atopoisomerase covalently bound to a 5′ terminus of one end or of bothends of the ds nucleotide sequence, and, unless indicated otherwise, thefirst and second (or other) ds nucleotide sequences can be the same orcan be different.

A method of the invention is additionally exemplified by contacting 1) afirst ds nucleotide sequence having a first end and a second end,wherein, at the first end, second end, or both ends, the first dsnucleotide sequence has a first topoisomerase covalently bound to the 5′terminus and a second topoisomerase covalently bound to the 3′ terminusof the first end, the second end, or both ends (i.e., one or both endscontain a topoisomerase-charged 5′ terminus and a topoisomerase-charged3′ terminus); and 2) at least a second ds nucleotide sequence, which,preferably, has, or can be made to have, hydroxyl groups at the 5′terminus and 3′ terminus of an end to be covalently linked to an end ofthe first ds nucleotide sequence containing the topoisomerases. Themethod also can be performed wherein either the 5′ terminus or 3′terminus of the end containing a topoisomerase-charged 3′ terminus ortopoisomerase-charged 5′ terminus, respectively, contains atopoisomerase recognition site, wherein the method further includescontacting the components with a topoisomerase that can effect itsactivity with respect to the topoisomerase recognition site. Such amethod of the invention can be performed using only a first dsnucleotide sequence and a second ds nucleotide sequence, or can includea third, fourth or more ds nucleotide sequence as desired, wherein theds nucleotide sequences are as defined for the first ds nucleotidesequence, the second ds nucleotide sequence, or a combination thereof. Afirst or second (or other) ds nucleotide sequence independently can, butneed not, have one or more topoisomerases covalently bound to a 5′terminus, 3′ terminus, or both 5′ and 3′ termini of the second end(i.e., the undefined end). Unless indicated otherwise, the first andsecond (or other) ds nucleotide sequences can be the same or can bedifferent.

The present invention further relates to a method of generating a dsrecombinant nucleic acid molecule covalently linked in both strandsby 1) amplifying a portion of a first ds nucleotide sequence using a PCRprimer pair, wherein at least one primer of the primer pair encodes acomplement of a topoisomerase recognition site, thereby producing anamplified first ds nucleotide sequence having a first end and a secondend, wherein the first end or second end or both has a topoisomeraserecognition site at or near the 3′ terminus; and 2) contacting a) theamplified first ds nucleotide sequence; b) at least a second dsnucleotide sequence having a first end and a second end, wherein thefirst end or second end or both has a topoisomerase recognition site, orcleavage product thereof, at or near the 3′ terminus and has, or can bemade to have, a hydroxyl group at the 5′ terminus of the same end; andc) a site specific topoisomerase, under conditions such that thetopoisomerase can cleave the end of the amplified first ds nucleotidesequence having a topoisomerase recognition site and the end (or ends)of the at least second ds nucleotide sequence having a topoisomeraserecognition site, and can effect its ligating activity. The PCR primerthat encodes a complement of a topoisomerase recognition site can have ahydroxyl group at its 5′ terminus, or the amplified first ds nucleotidesequence generated using the primer can be contacted with a phosphataseto generate a hydroxyl group at its 5′ terminus. The PCR primer encodingthe complement of a topoisomerase recognition site also can comprise anucleotide sequence at its 5′ terminus such that, upon cleavage by asite specific topoisomerase of a first ds nucleotide sequence amplifiedusing the primer, the ds nucleotide sequence contains a 5′ overhangingsequence, which is complementary to a 5′ overhang of a second (or other)ds nucleotide sequence to which the first ds nucleotide sequence is tobe covalently linked according to a method of the invention.

The present invention also relates to a method of generating a dsrecombinant nucleic acid molecule covalently linked in both strandsby 1) amplifying a portion of a first ds nucleotide sequence using a PCRprimer pair, wherein at least one primer of the primer pair encodes atopoisomerase recognition site, thereby producing an amplified first dsnucleotide sequence having a first end and a second end, wherein thefirst end, second end, or both ends have a topoisomerase recognitionsite at or near the 5′ terminus; and 2) contacting a) the amplifiedfirst ds nucleotide sequence; b) at least a second ds nucleotidesequence having a first end and a second end, wherein the first end,second end, or both ends have a topoisomerase recognition site at ornear the 5′ terminus and have, or can be made to have, a hydroxyl groupat the 3′ terminus of the same end; and c) at least one site specifictopoisomerase, under conditions such that the at least one topoisomerasecan cleave the first and/or second end of the amplified first dsnucleotide sequence having a topoisomerase recognition site and the end(or ends) of the at least second ds nucleotide sequence having atopoisomerase recognition site, and can effect its ligating activity.The amplified first ds nucleotide sequence generally has a hydroxylgroup at the 3′ terminus of the end containing the topoisomeraserecognition site, or can be modified to contain such a 3′ hydroxylgroup. The PCR primer encoding the topoisomerase recognition site canfurther comprise a nucleotide sequence at its 5′ terminus, i.e., 5′ tothe topoisomerase recognition site, such that, upon cleavage of theamplified first ds nucleotide sequence by a site specific topoisomerase,the ds nucleotide sequence contains a 3′ overhanging sequence, which iscomplementary to a 3′ overhanging sequence of a second (or other) dsnucleotide sequence to which the first ds nucleotide sequence is to becovalently linked according to a method of the invention.

The present invention further relates to a method of generating a dsrecombinant nucleic acid molecule covalently linked in both strandsby 1) amplifying a portion of a first ds nucleotide sequence using a PCRprimer pair, wherein at least one primer of the primer pair includes atopoisomerase recognition site and a nucleotide sequence complementaryto a topoisomerase recognition site, thereby producing an amplifiedfirst ds nucleotide sequence having a first end and a second end,wherein the amplified first ds nucleotide sequence has a topoisomeraserecognition site at or near the 5′ terminus and a topoisomeraserecognition site at or near the 3′ terminus of the first end, secondend, or both ends; and 2) contacting a) the amplified first dsnucleotide sequence; b) at least a second ds nucleotide sequence havinga first end and a second end, wherein the second ds nucleotide sequencehas, or can be made to have, a 5′ hydroxyl group and a 3′ hydroxyl groupat the first end, second end, or both ends; and c) at least two sitespecific topoisomerases, under conditions such that i) at least onetopoisomerase can cleave the topoisomerase recognition site at or nearthe 5′ terminus of the first and/or second end of the amplified first dsnucleotide sequence, and can effect its ligating activity, and ii) atleast one topoisomerase can cleave the topoisomerase recognition site ator near the 3′ terminus of the end of the amplified first ds nucleotidesequence, and can effect its ligating activity. Accordingly, the presentinvention provides a ds nucleotide sequence containing, at one or bothends, a topoisomerase recognition site at or near the 5′ terminus and atopoisomerase recognition site at or near the 3′ terminus. In addition,the invention provides such a ds nucleotide sequence, which istopoisomerase-charged at the 5′ terminus, the 3′ terminus, or bothtermini.

The present invention further relates to an isolated oligonucleotidecontaining a recognition site of a type IA site specific topoisomeraseand/or a nucleotide sequence complementary to a recognition site of atype IB site specific topoisomerase, such an oligonucleotide beinguseful, for example, as a primer for a primer extension reaction or asone of a primer pair for performing an amplification reaction such asPCR, as well as products generated by incubation with a topoisomerase.Such an oligonucleotide, which is referred to an oligonucleotide primer,can be one of a primer pair, which can be useful, for example, forgenerating a ds nucleic acid amplification product that contains, at oneend, a topoisomerase recognition site (e.g., a type IA or type IItopoisomerase recognition site) at or near the 5′ terminus and, at thesame end, a topoisomerase recognition site (e.g., a type IBtopoisomerase recognition site) at or near the 3′ terminus. Generally,the oligonucleotide primer is about 12 to 100 nucleotides in length, andusually about 15 to 50 nucleotides in length, particularly about 18 to30 nucleotides in length, wherein, when present, the nucleotide sequenceof the type IA topoisomerase recognition site and the nucleotidesequence complementary to the type IB topoisomerase recognition sitecan, but need not, be separated by at least one or a few (e.g., 1, 2, 3,4, 5, 6, 7, 8, etc.) nucleotides.

An oligonucleotide primer of the invention can further contain anucleotide sequence encoding (or complementary to) any other nucleotidesequence or peptide of interest, for example, one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, etc.) restriction endonuclease recognition sites, apeptide tag, and, if desired, one or more additional type IA, type II ortype IB topoisomerase recognition sites, thereby allowing selection ofone or more convenient or readily available topoisomerases forpracticing a method of the invention. The oligonucleotide primer canfurther comprise a nucleotide sequence at its 5′ terminus, i.e., 5′ tothe topoisomerase recognition site (e.g., type IA or type IItopoisomerase recognition site) or to the nucleotide sequencecomplementary to a type IB topoisomerase recognition site, such that,upon cleavage of the amplified first ds nucleotide sequence by a sitespecific topoisomerase, the ds nucleotide sequence contains a 3′ or 5′overhanging sequence, respectively, which is complementary to a 3′ or 5′overhanging sequence, respectively, of a second (or other) ds nucleotidesequence to which the first ds nucleotide sequence is to be covalentlylinked according to a method of the invention, or the oligonucleotideprimer can be designed such that, upon cleavage of an amplified dsnucleotide sequence generated therefrom, a blunt endtopoisomerase-charged ds nucleotide sequence is generated.

The present invention also provides a primer pair, which includes atleast one oligonucleotide primer as defined above, wherein one of theprimers is useful as a forward primer and the primer is useful as areverse primer in an amplification reaction. The first and/or secondprimer in such a primer pair can, but need not, include a type IAtopoisomerase recognition site, a nucleotide sequence complementary to atype IB topoisomerase recognition site, or both, and can include anyother nucleotide sequence of interest. In one embodiment, the primerpair includes at least two oligonucleotide primers of the invention,wherein one oligonucleotide primer is useful as a forward primer and thesecond oligonucleotide primer is useful as a reverse primer, such aprimer pair being useful, for example, for generating a ds nucleotidesequence amplification product having topoisomerase recognition sites atboth termini of both ends, wherein the type IA or type IB or bothtopoisomerase recognition sites at the termini are the same ordifferent. Accordingly, primer pairs of the invention include, forexample, a first primer encoding a type IA topoisomerase recognitionsite and a second primer encoding a nucleotide sequence complementary toa type IB topoisomerase recognition site; a first primer encoding a typeIA topoisomerase recognition site and a second primer encoding a type IAtopoisomerase recognition site, which can be the same or different asthat encoded by the first primer; a first primer encoding a nucleotidesequence complementary to a type IB topoisomerase recognition site and asecond primer encoding a nucleotide sequence complementary to a type IBtopoisomerase recognition site, which can be the same or different fromthat encoded by the first primer; a first primer encoding a type IAtopoisomerase recognition site and a second primer encoding a type IIrecognition site or a nucleotide sequence complementary thereto; a firstprimer encoding a nucleotide sequence complementary to a type IBtopoisomerase recognition site and a second primer encoding a type IItopoisomerase recognition site or a nucleotide sequence complementarythereto; a first primer encoding a type II topoisomerase recognitionsite or a nucleotide sequence complementary thereto and a second primerencoding a type II topoisomerase recognition site or a nucleotidesequence complementary thereto, which is the same or different from thetype II topoisomerase recognition site of the first primer. The presentinvention also provides kits containing one or more primer pairs of theinvention, for example, one or more of the primer pairs exemplifiedabove, or can contain three primers, for example, a first primerencoding a type IA topoisomerase recognition site, a second primerencoding a nucleotide sequence complementary to a type IB topoisomeraserecognition site, and a third primer encoding a type II topoisomeraserecognition site or a nucleotide sequence complementary thereto, such akit allowing a convenient means to generate a primer extension oramplification product that can be covalently linked according to amethod of the invention.

Accordingly, the present invention further relates to a ds nucleotidesequence, which has a first end and a second end, and which contains atopoisomerase recognition site (e.g., a type IA or type II topoisomeraserecognition site) at or near the 5′ terminus and a type IB topoisomeraserecognition site at or near the 3′ terminus of the first end, the secondend, or of both ends. In addition, the present invention provides a dsnucleotide sequence as defined above, except wherein the ds nucleotidesequence is a topoisomerase-charged molecule, comprising a stably boundtype IA topoisomerase or a type IB topoisomerase or both, at one or bothends, as desired.

In one embodiment, the first ds nucleotide sequence comprises or encodesan expressible nucleotide sequence such as a nucleotide sequenceencoding a polypeptide, an antisense nucleotide sequence, a ribozyme, atRNA (e.g., a suppressor tRNA), a triplexing nucleotide sequence or thelike, and the second (or other) ds nucleotide sequence comprises atranscription regulatory element such as a promoter (e.g., a GAL4promoter), an enhancer, a silencer, a translation start site, or apolyadenylation signal, or encodes a translation regulatory element suchas an initiator methionine, a STOP codon, a cell compartmentalizationdomain, a homology domain, or the like, or a combination thereof inoperative linkage. A second (or other) ds nucleotide sequence, which canbe an amplified second (or other) ds nucleotide sequence prepared as forthe amplified first ds nucleotide sequence, also can comprise adetectable label, for example, an enzyme, a substrate for an enzyme, afluorescent compound, a luminescent compound, a chemiluminescentcompound, a radionuclide, a paramagnetic compound, and biotin; or caninclude a tag, which can be an oligonucleotide tag or can be a peptidetag, for example, a polyhistidine tag, a V5 epitope, or a myc epitope.

In another embodiment, a method of the invention is performed using afirst ds nucleotide sequence that encodes a polypeptide, or a domainthereof, and a second (or other) ds nucleotide sequence that encodes atranscription activation domain or a DNA binding domain. Such a methodcan be used to generate covalently linked ds recombinant nucleic acidmolecules, covalently linked in one or both strands, that encodechimeric polypeptides useful for performing a two hybrid assay system,particularly a high throughput two hybrid assay. In still anotherembodiment, the first ds nucleotide sequences comprises a plurality ofnucleotide sequences, which can be a cDNA library, a combinatoriallibrary of nucleotide sequences, a variegated population of nucleotidesequences, or the like.

A method of the invention provides a means to generate a ds recombinantnucleic acid molecule, covalently linked in one or both strands, usefulfor site specific insertion into a target genomic DNA sequence. Thetarget genomic DNA sequence can be any genomic sequence, particularly agene, and preferably a gene for which some or all of the nucleotidesequence is known. The method can be performed utilizing two sets ofamplification primer pairs such as PCR primer pairs and a ds nucleotidesequence. The ds nucleotide sequence has a first end and a second endand generally encodes a polypeptide, for example, a selectable marker,wherein the ds nucleotide sequence comprises a topoisomerase recognitionsite or cleavage product thereof at the 3′ terminus of each end and,optionally, a hydroxyl group at the 5′ terminus of each end, andwherein, preferably, the 5′ termini comprise overhanging sequences,which are different from each other. Similarly, the ds nucleotidesequence can comprise a topoisomerase recognition site (or cleavageproduct thereof) at or near the 5′ terminus of one or both ends and,optionally, a hydroxyl group at the 3′ terminus of one or both end, andwherein one or both of the 3′ termini can comprise overhangingsequences, which can be the same as or different from each other; or the5′ terminus and 3′ terminus of one or both ends of the ds nucleotidesequence each can comprise a topoisomerase recognition site or cleavageproduct thereof (see FIGS. 4 and 5).

The two sets of PCR primer pairs generally are selected such that, inthe presence of an appropriate DNA polymerase such as Taq polymerase anda template comprising the sequences to be amplified, the primers amplifyportions of a genomic DNA sequence that are upstream (and adjacent to)and downstream (and adjacent to) of the target site for insertion of thepolypeptide (e.g., selectable marker). The sets of PCR primer pairs alsoare designed such that the amplification products contain atopoisomerase recognition site at least at the end to be covalentlylinked in one or both strands to the selectable marker, including at ornear the 5′ terminus, the 3′ terminus, or both termini, as appropriatefor the particular method of the invention being practiced. As such, thefirst PCR primer pair can include, for example, 1) a first primer, whichcomprises, in an orientation from 5′ to 3′, a nucleotide sequencecomplementary to a 5′ overhanging sequence of the end of the selectablemarker to which the amplification product is to be covalently linked, anucleotide sequence complementary to a topoisomerase recognition site,and a nucleotide sequence complementary to a 3′ sequence of a targetgenomic DNA sequence; and 2) a second primer, which comprises anucleotide sequence of the target genomic DNA upstream of the 3′sequence to which the first primer is complementary. The second PCRprimer pair includes 1) a first primer, which comprises, from 5′ to 3′,a nucleotide sequence complementary to the 5′ overhanging sequence ofthe end of the selectable marker to which it is to be covalently linked,a nucleotide sequence complementary to a topoisomerase recognition site,and a nucleotide sequence of a 5′ sequence of a target genomic DNAsequence, wherein the 5′ sequence of the target genomic DNA isdownstream of the 3′ sequence of the target genomic DNA to which thefirst primer of the first PCR primer pair is complementary; and 2) asecond primer, which comprises a nucleotide sequence complementary to a3′ sequence of the target genomic DNA that is downstream of the 5′sequence of the target genomic DNA contained in the first primer.

Upon contact of the ds nucleotide sequence comprising the selectablemarker, the PCR amplification products, and at least one topoisomerase,a ds recombinant nucleic acid molecule, covalently linked in one or bothstrands, is generated according to a method of the invention. Thegenerated ds recombinant nucleic acid molecule is useful for performinghomologous recombination in a genome, for example, to knock-out thefunction of a gene in a cell, or to confer a novel phenotype on the cellcontaining the generated ds recombinant nucleic acid molecule. Themethod can further be used to produce a transgenic non-human organismhaving the generated recombinant nucleic acid molecule stably maintainedin its genome.

The present invention also relates to compositions prepared according tothe methods of the invention, and to compositions useful for practicingthe methods. Such compositions can include one or more reactants used inthe methods of the invention and/or one or more ds recombinant nucleicacid molecules produced according to a method of the invention. Suchcompositions can include, for example, one or more topoisomerase-chargeds nucleotide sequences; one or more primers useful for preparing a dsnucleotide sequence containing a topoisomerase recognition site at oneor both termini of one or both ends of an amplification product preparedusing these primers; one or more topoisomerases; one or more substrateds nucleotide sequences, including, for example, nucleotide sequencesencoding tags, markers, regulatory elements, or the like; one or more dsrecombinant nucleic acid molecules covalently linked in one or bothstrands, produced according to a method of the invention; one or morecells containing or useful for containing a ds nucleotide sequence,primer, or recombinant nucleic acid molecule as disclosed herein; one ormore polymerases for performing a primer extension or amplificationreaction; one or more reaction buffers; and the like. In one embodiment,a composition of the invention comprises two or more differenttopoisomerase-charged ds nucleotide sequences. The composition canfurther comprise at least one topoisomerase. A composition of theinvention also can comprise a site specific topoisomerase and a dsrecombinant nucleic acid molecule covalently linked in one or bothstrands, wherein the recombinant nucleic acid molecule contains at leastone topoisomerase recognition site for the site specific topoisomerasein each strand. The topoisomerase recognition site in one strand can beany distance from a topoisomerase recognition site in the complementarystrand, for example, wherein a topoisomerase recognition site in onestrand is within about 100 nucleotides of a topoisomerase recognitionsite in the complementary strand, or wherein the recognition sites arewithin about 50 nucleotides of each other, or within about 20nucleotides of each other, or less.

Methods of the invention also can be used to link at least one end of adouble stranded nucleic acid molecule (e.g., DNA or RNA) to at least oneend of a single stranded nucleic acid molecule (e.g., DNA or RNA).Furthermore, the methods of the invention can be used to link at leastone end of a single stranded nucleic acid molecule (e.g., DNA or RNA) toat least one end of a second (or other) single stranded nucleic acidmolecule (e.g., DNA or RNA). In appropriate circumstances, the methodsof the invention can be used to circularize nucleic acid molecules,including to concatenate and circularize nucleic acid molecules. Thus,one or more ds nucleotide sequences disclosed herein as useful in anaspect or embodiment of the invention can be replaced with one or moresingle stranded nucleotide sequences. The invention further includescompositions used in such methods and nucleic acid molecules produced bysuch methods. Thus, for example, the invention includes single-strandednucleic acid molecules to which a site-specific topoisomerase (e.g., atype IA topoisomerase, a type IB topoisomerase, a type II topoisomerase,etc.) is attached to the 5′ or 3′ terminus. Methods for joining singlestranded nucleic acid molecules to other single stranded nucleic acidmolecules are described, for example, in Internatl. Publ. No. WO00/56878, which is incorporated herein by reference.

The present invention provides methods for joining DNA molecules to RNAmolecules, as well as compositions used in such methods and nucleic acidmolecules produced by such methods. Thus, nucleotide sequences of theinvention can comprise, for example, DNA (e.g., cDNA, genomic DNA,plasmid DNA, synthetic DNA, etc.) or RNA (e.g., mRNA, rRNA, tRNA,synthetic RNA, ribozymes, etc.). Examples of such methods are set out,for example, in FIG. 8 and in Internatl. Publ. No. WO 98/56943, which isincorporated herein by reference.

The present invention also relates to a kit, which contains componentsthat can be useful for practicing a method of the invention. A kit ofthe invention can contain, for example, one or moretopoisomerase-charged ds nucleotide sequence substrates, which caninclude one or more control nucleotide sequences that can be useful, forexample, to test the accuracy or fidelity of the components of the kit;one or more topoisomerases; one or more primers, which can comprise atopoisomerase recognition site, a nucleotide sequence complementary to atopoisomerase recognition site, or both; one or more cells, which cancontain or be useful for containing a nucleotide sequence of the kit ora nucleic acid molecule generated using the kit; one or more reagents,polymers, buffers, or the like, for performing a method using the kit;instructions for performing a method using the kit, for example,instructions for covalently linking one strand of first nucleotidesequence to one strand of at least a second nucleotide sequence, eitheror both of which can be single stranded or double stranded nucleotidesequences, or instructions for covalently linking both strands of afirst ds nucleotide sequence to both strands of at least a second dsnucleotide sequence; and the like.

In one aspect, a kit of the invention contains a ds nucleotide sequencehaving a first end and a second end and encoding a polypeptide, whichcan be expressed, for example, a selectable marker, wherein the dsnucleotide sequence comprises a topoisomerase recognition site orcleavage product thereof at the 3′ terminus of one or both ends.Optionally, the ds nucleotide sequence contains a hydroxyl group at the5′ terminus of one or both of the other ends, preferably at the endcontaining the topoisomerase recognition site or that istopoisomerase-charged. In particular embodiments, one or both 5′ terminicomprise overhanging sequences, which can be the same or can bedifferent from each other.

A kit of the invention also can contain a ds nucleotide sequence havinga first end and a second end, and encoding a polypeptide to beexpressed, for example, a selectable marker, wherein the ds nucleotidesequence comprises a topoisomerase recognition site or cleavage productthereof at the 5′ terminus of one or both ends. Optionally, the dsnucleotide sequence contains a hydroxyl group at the 3′ terminus of oneor both ends, and preferably, one or both 3′ termini compriseoverhanging sequences, which can be the same or can be different fromeach other. In addition, a kit of the invention can contain a dsnucleotide sequence having a first end and a second end, and encoding apolypeptide to be expressed, for example, a selectable marker, whereinthe ds nucleotide sequence comprises a topoisomerase recognition site orcleavage product thereof at the 5′ terminus and the 3′ terminus of oneor both ends. As such, it should be recognized that a kit of theinvention can include any of various combinations of such ds nucleotidesequences comprising one or more topoisomerase recognition sites ortopoisomerase-charged ds nucleotide sequences, including ds nucleotidesequences having a topoisomerase recognition site at a terminus or bothtermini of one or both ends and that is topoisomerase-charged at one ormore termini.

A kit of the invention also can contain a ds nucleotide sequencecomprising a regulatory element or other nucleotide sequence, forexample, a coding sequence, and a topoisomerase recognition site orcleavage product thereof at a 3′ terminus of at least a first end and,optionally, a hydroxyl group at the 5′ terminus of an end containing therecognition site; or comprising a topoisomerase recognition site orcleavage product thereof at a 5′ terminus of at least a first end, and,optionally, a hydroxyl group at the 3′ terminus of the end containingthe recognition site; or comprising a topoisomerase recognition site atthe 5′ terminus and 3′ terminus of at least a first end. Preferably, thekit contains a variety of upstream regulatory elements, a variety ofdownstream regulatory elements, a variety of elements useful detectingor identifying a molecule containing the element, and combinationsthereof. For example, the kit can contain a variety of gene promoterelements, which are constitutively active or inducible in one or a fewor many different types of cells, elements that permit or facilitateribosome binding such as an internal ribosome entry site, an elementencoding a Kozak sequence or an initiator methionine, or the like. Inaddition, or alternatively, the kit can contain a variety of downstreamregulatory elements such a polyadenylation signal sequences, sequencesthat terminate transcription or translation, or the like; and also cancontain enhancers, silencers, and the like. Similarly, the kit cancontain elements encoding detectable markers such as epitope tags, orthe like. Preferably, the kit contains a variety of such elements, eachof which contains at least one topoisomerase recognition site. Morepreferably, the elements further contain an overhanging sequence suchthat they can be operatively covalently linked to each other or to a dsnucleotide sequence encoding a polypeptide such as a selectable markeraccording to a method of the invention.

Optionally, a kit of the invention can contain element specific primers,which can be used to amplify a construct containing one of the varietyof elements included in the kit. Where the kit contains such primers,the ds nucleotide sequences comprising the regulatory or other elementhas a nucleotide sequence that can be specifically bound by the primersuch that extension of the primer through and including the regulatoryelement can be effected. In particular, the kit can contain elementspecific forward and reverse primers, which can be combined to produce aprimer pair useful for amplifying, for example, a recombinant nucleicacid molecule containing a particular 5′ regulatory element and aparticular 3′ regulatory element of the kit. Such a primer pair canselectively amplify a desired functional ds recombinant nucleic acidmolecule covalently linked in both strands generated according to amethod of the invention, but does not amplify partial reaction products.

In another embodiment, a kit of the invention contains a first dsnucleotide sequence, which has a first end and a second end, contains atopoisomerase recognition site, or cleavage product thereof, at one orboth 3′ termini, and encodes a transcription activation domain; and asecond ds nucleotide sequence, which has a first end and a second end,contains a topoisomerase recognition site, or cleavage product thereof,at one or both 3′ termini, and encodes a DNA binding domain; or containsa first ds nucleotide sequence, which has a first end and a second end,contains a topoisomerase recognition site, or cleavage product thereof,at one or both 5′ termini, and encodes a transcription activationdomain; and a second ds nucleotide sequence, which has a first end and asecond end, and optionally contains a topoisomerase recognition site, orcleavage product thereof, at one or both 5′ termini, and encodes a DNAbinding domain. A kit of the invention also can contain a first dsnucleotide sequence, which has a first end and a second end, and encodesa transcription activation domain, and a second ds nucleotide sequence,which has a first end and a second end, and encodes a DNA bindingdomain, wherein at least the first ds nucleotide sequence or the secondds nucleotide sequence contains a topoisomerase recognition site; orcleavage product thereof, at a 5′ terminus and a 3′ terminus of at leastone end, and wherein the other ds nucleotide contains a 3′ hydroxyl and5′ hydroxyl at the end to be covalently linked to the end of the dsnucleotide sequence comprising the recognition sites.

Such a kit is useful, for example, for generating a ds recombinantnucleic acid molecule covalently linked in both strands, or a dsrecombinant nucleic acid molecule covalently linked in one strand,encoding chimeric polypeptides for performing a two hybrid assay. Thekit can further contain a primer pair, which can amplify a nucleotidesequence to be operatively linked to the first or second ds nucleotidesequence, wherein at least one primer of the primer pair comprises atopoisomerase recognition site, a complement of a topoisomeraserecognition site, or both. Preferably, an amplification productgenerated using such a primer pair contains, following cleavage by asite-specific topoisomerase, a 3′ or 5′ overhanging sequence that iscomplementary to the first or second ds nucleotide sequence to which itis to be covalently linked. Such a kit can facilitate the generation ofrecombinant polynucleotides that comprise a first or second nucleotidesequence of the kit and encode a chimeric polypeptide useful forperforming a two hybrid assay.

In another embodiment, a kit of the invention contains a first dsnucleotide sequence having a first end and a second end, each end havinga 5′ terminus and a 3′ terminus; and instructions for using atopoisomerase to covalently linking the 5′ terminus and 3′ terminus ofat least one of the first end and the second end to a 5′ terminus and a3′ terminus of a second ds nucleotide sequence. Such a kit also cancontain a second (or more) ds nucleotide sequence, to which the first dsnucleotide sequence can be covalently linked in both strands accordingto the instructions. In addition, the kit can contain a topoisomerase,for example, a type IB topoisomerase such as a Vaccinia type IBtopoisomerase. The first ds nucleotide sequence is such a kit cancontain at least one topoisomerase recognition site at or near the 5′terminus or 3′ terminus of the first end or second end or both ends, forexample, a type IB topoisomerase recognition site at or near a 3′terminus of one or both ends; or can have a topoisomerase bound to atleast one terminus of the first end or second or both ends, for example,a type IB topoisomerase bound to a 3′ terminus of the first end orsecond end or both.

The present invention further relates to a method of generating dsrecombinant RNA molecules. Such a method can be performed, for example,by 1) contacting a) a first topoisomerase-charged ds nucleotide sequencehaving a first end and a second end, each end having a 5′ terminus and a3′ terminus, wherein each of said first end and said second end includesa topoisomerase bound at the 3′ terminus and a hydroxyl group at the 5′terminus; and b) at least second topoisomerase-charged ds nucleotidesequence having a first end and a second end, each end having a 5′terminus and a 3′ terminus, wherein said at least secondtopoisomerase-charged ds nucleotide sequence comprises a promoter for anRNA polymerase, and wherein said first end or said second end or bothhas a topoisomerase bound at the 3′ terminus and a hydroxyl group at the5′ terminus, under conditions such that an end of a first ds nucleotidesequence having a topoisomerase covalently bound thereto contacts an endof the at least second ds nucleotide sequence having a topoisomerasecovalently bound thereto, thereby generating a ds recombinant nucleicacid molecule covalently linked in both strands; and 2) contacting theds recombinant nucleic acid molecule covalently linked in both strandswith an RNA polymerase specific for the promoter, under conditionssuitable for transcription of RNA by the RNA polymerase andhybridization of transcribed complementary RNA molecules, therebygenerating ds RNA molecules (see Appendix A, which is incorporatedherein by reference; see, e.g., pages A45-A98). According to the presentmethod, the RNA polymerase promoter any such promoter, including, forexample, a viral RNA polymerase promoter such as a T3, promoter, a T7promoter, an SP6 promoter, and the like. In many instances, the promoteris a promoter that is suitable for use in an in vitro transcriptionsystem Accordingly, ds nucleic acid molecules having an RNA polymerasepromoter linked in one or both strands also are provided, as are ds RNAmolecules generated therefrom.

The invention thus provides methods for generating nucleic acidmolecules that allow for the expression and/or generation of ds RNAmolecules. For example, using methods of the invention, two nucleic acidsegments can be connected to each other, wherein one of the nucleic acidsegments comprises nucleic acid which functions as a promoter and theother nucleic acid segment comprises nucleic acids which can betranscribed to form at least one strand of a ds RNA molecule. Thus, dsRNA molecules can be prepared by transcription (e.g., in vitrotranscription) of a nucleic molecule (e.g., a DNA molecule) whichencodes one strand or both strands of the ds RNA molecule. When thenucleic acid molecule encodes both strands of the ds RNA molecule, thesestrands can be produced as a single transcript or as separatetranscripts. When both strands are produced as a single transcript, thetwo complementary portions of the transcript can be connected by alinker which forms a single stranded region when the complementaryregions anneal to each other. This linker region can be of any suitablelength (e.g., three, four, five, six, seven, eight, nine, ten, etc.nucleotides).

Further nucleic acid molecules that can be used to express doublestranded RNA molecules can comprise nucleic acid segments comprising apromoter connected to nucleic acid that is to be transcribed, whereinthese two segments are linked in one strand or in both strands. In manyinstances, when nucleic acid segments are joined in only one strand,either the nucleic acid segment which comprises a promoter activity orthe other nucleic acid segment will contain a bound topoisomerasemolecule. As described elsewhere herein, in many instances, thistopoisomerase molecule will be bound to a 3′ terminus of thetopoisomerase adapted nucleic acid segment. In instances where only oneend of termini that are joined contains a bound topoisomerase, the otherend that is involved in the joining reaction can, but need not, containa topoisomerase recognition sequence.

The invention further includes methods for generating ds RNA moleculeemploying nucleic acid molecules of the invention. In one aspect, theinvention include performing in vitro transcription on nucleic acidmolecules described herein. In vitro transcription reactions are knownin the art and are described, for example, in U.S. Pat. No. 5,256,555(which is incorporated herein by reference), and elsewhere herein.

A method of producing ds RNA molecules as disclosed herein can furtherinclude a step of contacting the ds RNA molecules with an enzyme thatcleaves the ds RNA molecules, and particularly with an enzyme thatcleaves the ds RNA molecule into oligoribonucleotides of a desiredlength (see Appendix A, e.g., pages A1 A44). Thus in one embodiment, themethod includes contacting the ds RNA molecules with an enzyme thatcleaves the ds RNA molecules into ds oligoribonucleotide moleculesconsisting of about 21 (e.g., 19, 20, 21, 22, or 23) nucleotides in eachstrand, thereby generating diced ds oligoribonucleotide molecules. Theenzyme used according to the present method can be any enzyme thatcleaves ds RNA molecules, including, for example, a dicer enzyme such asthe BLOCK-iT™ dicer enzyme (Invitrogen Corp.; Carlsbad Calif.).

In one aspect, the generated diced ds oligoribonucleotide moleculesconsist of two strands of 19 to 21 nucleotides each. In another aspect,the generated diced ds oligoribonucleotide molecules consist of twostrands of 19 to 21 nucleotides each, wherein the ds oligoribonucleotidemolecules further contains an at least one (e.g., 1, 2, 3, 4, etc.)nucleotide overhang at one or both ends (e.g., 2 nucleotide overhangs onboth ends). In still another aspect, the diced ds oligoribonucleotidemolecules have short interfering RNA (siRNA); i.e., the method generatesdiced siRNA (d-siRNA) molecules.

In many instances, ds oligoribonucleotides will be molecules that areabout 21 to 23 nucleotides in length with two nucleotide overhangs oneach end (e.g., two nucleotide 3′ overhangs). Thus, when a dsoligoribonucleotide is 23 nucleotides in length with two nucleotideoverhangs on each end, it will typically be composed of two RNA strands,each of which is each 21 nucleotides in length.

A method of generating diced ds oligoribonucleotide molecules asdisclosed herein can further include a step of isolating the diced dsoligoribonucleotide molecules, thereby obtaining isolated diced dsoligoribonucleotide molecules. Such a method can be performed using anyconvenient methods for isolating such oligoribonucleotides, including,for example, chromatographic (e.g., gel filtration chromatography suchas HPLC, affinity chromatography, or electrophoresis) using readilyavailable reagents or commercially available kits. As such, a method ofthe invention provides a means to obtain, for example, isolated d-siRNAmolecules, which can be used to reduce or inhibit gene expression in acell. Accordingly, the present invention also relates to a method ofreducing or inhibiting expression of a target gene in a cell bycontacting cells including the target cell with d-siRNA moleculesobtained according to a method of the invention, whereby the d-siRNAreduces or inhibits expression of the target gene in the cell.

The present invention further relates to compositions produced accordingto the methods of the invention, including ds RNA molecules,compositions containing diced ds oligoribonucleotide molecules and/ord-siRNA molecules, and isolated diced ds oligoribonucleotide moleculesand/or d-siRNA molecules. The present invention also provides kits forpracticing the invention methods, including, for example, kitscontaining reagents for generating a ds RNA molecule having an RNApolymerase promoter at one or both ends, reagents for dicing such a dsRNA molecule and/or for isolating diced ds oligoribonucleotidemolecules, and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict generating a covalently linked double strandednucleotide sequence containing an element on each end according to amethod of the invention. “PCR” indicates polymerase chain reaction;“TOPO” indicates topoisomerase; topoisomerase shown as circle attachedto sequence; “P1” and “P2” indicate PCR primers. Topoisomeraserecognition site is indicated in bold print.

FIGS. 2A to 2C show the ends of PCR products representing acytomegalovirus promoter element (“CMV”), a green fluorescent proteinelement (“GFP”), and a bovine growth hormone polyadenylation signal(“BGH”) element. Primers used to construct the PCR products of FIGS. 2A,2B and 2C are indicated by an “F” number (see Table 1). The portion ofone or both ends including the topoisomerase recognition site (CCCTT) isshown. Bold print indicates overhanging sequences. In FIGS. 2A and 2B,one (FIG. 2B) or both (FIG. 2A) of the overhang sequences arepalindromic in nature. Sequences are shown in conventional orientation,with the top strand in a 5′ to 3′ orientation from left to right, andthe bottom strand in a 3′ to 5′ orientation from left to right. Numberin parentheses above or below sequence indicates SEQ ID NO:.

FIGS. 3A and 3B show constructs (FIG. 3A) and results (FIG. 3B) ofexperiments examining the ability to use ds recombinant nucleic acidmolecule covalently linked in both strands that encode polypeptides forperforming a two hybrid assay.

FIG. 3A shows the amount of each construct used for transfection. A “p”preceding an amount or volume of reactant indicates plasmid form, “1”indicates linear form, and “PCR” indicates PCR amplification reactionmixture.

FIG. 3B shows the level of β-galactosidase activity (“LacZ activity”)associated with each transfected sample. Increased LacZ activity isindicative of a positive interaction.

FIGS. 4A to 4F represent various embodiments of the composition and ofthe invention for generating a ds recombinant nucleic acid moleculecovalently linked in one Topoisomerase is shown as a solid circle, andis either attached to a terminus of a substrate ds nucleotide sequenceor is released following a linking reaction.

FIGS. 5A to 5D illustrate various embodiments of compositions andmethods of the invention for generating a covalently linked dsrecombinant nucleic acid molecule. Topoisomerase is shown as a solidcircle, and is either attached to a terminus of a substrate dsnucleotide sequence or is released following a linking reaction. Asillustrated, the substrate ds nucleotide sequences have 5′ overhangs,although they similarly can have 3′ overhangs or can be blunt ended. Inaddition, while the illustrated ds nucleotide sequences are shown havingthe topoisomerases bound thereto (topoisomerase-charged), one or more ofthe termini shown as having a topoisomerase bound thereto also can havea topoisomerase recognition site (i.e., one or more termini containing atopoisomerase recognition site), in which case the joining reactionwould further require addition of one or more site specifictopoisomerases, as appropriate.

FIG. 5A shows a first ds nucleotide sequence having a topoisomeraselinked to each of the 5′ terminus and 3′ terminus of one end, andfurther shows linkage of the first ds nucleotide sequence to a second dsnucleotide sequence.

FIG. 5B shows a first ds nucleotide sequence having a topoisomerasebound to the 3′ terminus of one end, and a second ds nucleotide sequencehaving a topoisomerase bound to the 3′ terminus of one end, and furthershows a covalently linked ds recombinant nucleic acid molecule generateddue to contacting the ends containing the topoisomerase-chargedsubstrate ds nucleotide sequences.

FIG. 5C shows a first ds nucleotide sequence having a topoisomerasebound to the 5′ terminus of one end, and a second ds nucleotide sequencehaving a topoisomerase bound to the 5′ terminus of one end, and furthershows a covalently linked ds recombinant nucleic acid molecule generateddue to contacting the ends containing the topoisomerase-chargedsubstrate ds nucleotide sequences.

FIG. 5D shows a ds nucleotide sequence having a topoisomerase linked toeach of the 5′ terminus and 3′ terminus of both ends, and further showslinkage of the topoisomerase-charged ds nucleotide sequence to two dsnucleotide sequences, one at each end. The topoisomerases at each of the5′ termini and/or at each of the 3′ termini can be the same ordifferent.

FIG. 6 illustrates the generation of an expressible ds recombinantnucleic acid molecule and amplification of the expressible dsrecombinant nucleic acid molecule. Topoisomerase is shown as a solidcircle, and is either attached to a terminus of a substrate dsnucleotide sequence or is released following a linking reaction. Theexpressible ds recombinant nucleic acid molecule is generated from threeds nucleotide sequences, including a nucleotide sequence comprising apromoter, a nucleotide sequence comprising a coding sequence, and anucleotide sequence comprising a polyadenylation signal. Generation ofthe nucleic acid molecule can be facilitated by the incorporation ofcomplementary 5′ and/or 3′ overhanging sequences at the ends of the dsnucleotides sequences to be joined. The expressible ds recombinantnucleic acid molecule is generated by contacting a first ds nucleotidesequence having a type IA topoisomerase at a 5′ terminus of a first endand a type IB topoisomerase at a 3′ terminus of a second end, with asecond ds nucleotide sequence and a third double stranded nucleotidesequence. The expressible ds recombinant nucleic acid molecule isamplified using a first primer that hybridizes to the second dsrecombinant nucleic acid molecule upstream of the promoter, and a secondprimer that hybridizes to the third ds recombinant nucleic acid moleculedownstream of the polyadenylation signal.

FIG. 7 shows one example of a process for preparing a double strandednucleic acid molecule which contains a topoisomerase (e.g., a type IAtopoisomerase) bound to the 5′ terminus of one end of the molecule,wherein the same end of the molecule further comprise a 3′ overhang (see(4) in this figure).

FIG. 8 shows two embodiments of the invention in which a single strandedor double stranded DNA nucleotide sequence is joined with a singlestranded RNA nucleotide sequence.

FIG. 9 provides a schematic outline exemplifying methods of theinvention. In the first step, nucleotide sequences to be assembled aregenerated using an amplification method such as PCR. In the second step,the nucleotide sequences generated in the first step are assembled usinga method of the invention (e.g., a method utilizing a topoisomerase tocovalently link at least one strand of one nucleotide sequence to atleast one strand of a second (or other) nucleotide sequence). In thethird step as exemplified, assembled nucleic acid molecules (i.e.,recombinant nucleic molecules) generated in the second step can be useddirectly or can be amplified, then used for any purpose as disclosedherein or otherwise desired.

FIG. 10 shows a diagram of the iRNA process and pathway.

FIG. 11 shows an example of expected results of a lacZ dicing reaction.

FIG. 12 shows a flow diagram illustrating the d-siRNA purificationprocess.

FIG. 13 shows an example of expected results following purification oflacZ d-siRNA.

FIG. 14 shows ds-iRNA inhibition of luciferase and β-galactosidase aspercent of control versus transfection condition.

FIG. 15 shows inhibition of expression of lamin A/C expression usingd-siRNA.

FIG. 16 illustrates the major steps necessary to generate dsRNA usingthe BLOCK-iT™ RNAi TOPO® Transcription System.

FIG. 17 shows TOPO® linking to a PCR product.

FIG. 18 shows the RNAi process and pathway.

FIG. 19 is a diagram of the BLOCK-iT T7-TOPO linker. FIG. 19 shows theT7 promoter (SEQ ID NO: 61) and its complement (SEQ ID NO: 62).

FIG. 20 shows an analysis of an annealing reaction of GFP and luciferasedsRNA samples.

FIG. 21 is a vector map of pcDNA™ 1.2/V5-GW/lacZ.

FIG. 22 shows fractionation of double-stranded RNA using differentethanol concentrations.

FIGS. 23A-23C show: 23A) gel analysis results of crude lacZ siRNA, siRNApurified using the two-column protocol, various fractions of thesingle-column purification protocol, as well as chemically synthesizedsiRNA analyzed on a 4% E-Gel, which were used for functional testing;23B) measurements of luciferase activities after transfection of cellswith lacZ siRNA; 23C) measurements of β-galactosidase activities aftertransfection of cells with lacZ siRNA

FIGS. 24A-24B show purification of siRNA generated with Dicer andRNaseIII.

FIG. 25 shows functional testing of siRNA preparations with F1pIn293-luccells. Relative luciferase activity was measured for siRNA samples.

FIGS. 26A-26B show functional testing of siRNA preparations withGripTite™ 293 MSR cells. 26A) Beta-galactosidase assay: Effect of lucsiRNA and lacZ siRNA generated with Dicer and RNaseIII enzyme onβ-galactosidase activity. 26B) Luciferase assay: Effect of luc siRNA andlacZ siRNA generated with Dicer and RNaseIII enzyme on luciferaseactivity.

FIGS. 27A-27B show determination of column capacity and recoveryefficiency. 27A) Recovery of tRNA after binding to the column matrixwith a single 100-μl or two 50-μl elutions. 27B) Recovery of a 1-kbdsRNA fragment after binding to the column matrix with a single 100-μlor two 50-μl elutions.

FIGS. 28A-28B show clean-up of long dsRNA and tRNA. 28A) Clean-up of100-, 500-, and 1000-bp fragments of dsRNA. 28B) Clean-up of yeast tRNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of using one or moretopoisomerases to generate a recombinant nucleic acid molecule from twoor more nucleotide sequences. In a first aspect, the invention providesa method for generating a ds recombinant nucleic acid molecule that iscovalently linked in one strand. Such a method is directed to linking afirst and at least a second nucleotide sequence with at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) topoisomerase (e.g., a type IA,type IB, and/or type II topoisomerase) such that one strand, but notboth strands, is covalently linked (see, for example, FIG. 4). In asecond aspect, the invention provides a method for generating a dsrecombinant nucleic acid molecule covalently linked in both strands.Such a method is directed to linking a first and at least a secondnucleotide sequence with at least one topoisomerase, such that ligatedends are covalently linked in both strands (i.e., the ds recombinantnucleic acid molecule contain no nicks at the positions where ends wereligated; see, for example, FIG. 5). In a third aspect, the inventionprovides a method for generating a recombinant nucleic acid moleculecovalently linked in one strand, wherein the substrate nucleotidesequences linked according to the method include at least one singlestranded nucleotide sequence, which can be covalently linked to a second(or more) single stranded nucleotide sequence or to a ds nucleotidesequence (see, for example, FIG. 8).

Covalently linked recombinant nucleic acid molecules assembled using themethods of the invention can be used directly, or can be amplified,first, then used for any number of procedures as exemplified herein orotherwise known in the art. As disclosed herein, covalently linkedrecombinant nucleic acid molecules can be generated from nucleotidesequence in any of a number of ways (see, for example, FIG. 9). Thenucleotide sequences useful in practicing the methods can be obtainedusing any of various well known methods, including, for example, bychemical synthesis, by isolation of restriction fragments or othercleavage products of genomic DNA, or by isolation of RNA, which can beused directly or converted to a cDNA using a reverse transcriptionmethod. Where the nucleotide sequences to be used according to a methodof the invention lack one or more termini or regions suitable forgeneration of a recombinant nucleic acid molecule, the termini and/orregions can be added to the nucleotide sequence, for example, by anamplification reaction such as PCR, wherein one or both primers encodethe desired sequence or a complement thereof (e.g., a topoisomeraserecognition site, an overhanging sequence, etc) or by ligating one ormore (e.g., one, two, three, four, etc.) adapter linkers, which cancontain, for example, one or more topoisomerase recognition sites, orthe nucleotide sequence can be modified using, for example, a methodsuch as site directed mutagenesis to convert, for example, a sequenceresembling a topoisomerase site to an actual topoisomerase recognitionsite. The nucleotide sequences having suitable termini and/or regionsthen can be assembled using methods of the invention as disclosedherein. The covalently linked recombinant nucleic acid moleculegenerated therefrom then can be amplified in vivo or in vitro, then usedin any number of methods or processes, including those exemplifiedherein or otherwise known in the art. The covalently linked recombinantnucleic acid molecules also can be used directly for applications suchas in vitro transcription/translation, recombinational cloning, or fortransforming or transfecting cells. Accordingly, the present inventionprovides versatile methods for manipulating nucleotide sequences and forgenerating covalently linked recombinant nucleic acid molecules havingdesirable characteristic, and further provides compositions containingsuch nucleotide sequences and/or recombinant nucleic acid molecules, aswell as methods of using the covalently linked recombinant nucleic acidmolecules.

A method for generating a ds recombinant nucleic acid moleculecovalently linked in one strand can be performed by contacting a firstds nucleotide sequence which has a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite), or a cleavage product thereof, at a 5′ or 3′ terminus, with asecond (or other) ds nucleotide sequence, and optionally, atopoisomerase (e.g., a type IA, type IB, and/or type II topoisomerase),such that the second nucleotide sequence can be covalently attached tothe first nucleotide sequence. As disclosed herein, the methods of theinvention can be performed using any number of nucleotide sequences,typically ds nucleotide sequences wherein at least one of the nucleotidesequences has a site-specific topoisomerase recognition site (e.g., atype IA, or type II topoisomerase), or cleavage product thereof, at oneor both 5′ termini (see, for example, FIGS. 4A-4F).

A method for generating a ds recombinant nucleic acid moleculecovalently linked in both strands can be performed, for example, bycontacting a first ds nucleotide sequence having a first end and asecond end, wherein, at the first end or second end or both, the firstds nucleotide sequence has a topoisomerase recognition site (or cleavageproduct thereof) at or near the 3′ terminus; at least a second dsnucleotide sequence having a first end and a second end, wherein, at thefirst end or second end or both, the at least second double strandednucleotide sequence has a topoisomerase recognition site (or cleavageproduct thereof) at or near a 3′ terminus; and at least one sitespecific topoisomerase (e.g., a type IA and/or a type IB topoisomerase),under conditions such that all components are in contact and thetopoisomerase can effect its activity. A covalently linked dsrecombinant nucleic acid generated according to a method of this aspectof the invention is characterized, in part, in that it does not containa nick in either strand at the position where the ds nucleotidesequences are joined. In one embodiment, the method is performed bycontacting a first ds nucleotide sequence and a second (or other) dsnucleotide sequence, each of which has a topoisomerase recognition site,or a cleavage product thereof, at the 3′ termini or at the 5′ termini oftwo ends to be covalently linked. In another embodiment, the method isperformed by contacting a first ds nucleotide sequence having atopoisomerase recognition site, or cleavage product thereof, at the 5′terminus and the 3′ terminus of at least one end, and a second (orother) ds nucleotide sequence having a 3′ hydroxyl group and a 5′hydroxyl group at the end to be linked to the end of the first dsnucleotide sequence containing the recognition sites. As disclosedherein, the methods can be performed using any number of ds nucleotidesequences having various combinations of termini and ends (see, forexample, FIG. 5A-5D).

Topoisomerases are categorized as type I, including type IA and type IBtopoisomerases, which cleave a single strand of a double strandednucleic acid molecule, and type II topoisomerases (gyrases), whichcleave both strands of a nucleic acid molecule. Type IA and IBtopoisomerases cleave one strand of a ds nucleotide sequence. Cleavageof a ds nucleotide sequence by type IA topoisomerases generates a 5′phosphate and a 3′ hydroxyl at the cleavage site, with the type IAtopoisomerase covalently binding to the 5′ terminus of a cleaved strand.In comparison, cleavage of a ds nucleotide sequence by type IBtopoisomerases generates a 3′ phosphate and a 5′ hydroxyl at thecleavage site, with the type IB topoisomerase covalently binding to the3′ terminus of a cleaved strand. As disclosed herein, type I and type IItopoisomerases, as well as catalytic domains and mutant forms thereof,are useful for generating ds recombinant nucleic acid moleculescovalently linked in both strands according to a method of theinvention.

Type IA topoisomerases include E. coli topoisomerase I, E. colitopoisomerase III, eukaryotic topoisomerase II, archeal reverse gyrase,yeast topoisomerase III, Drosophila topoisomerase III, humantopoisomerase III, Streptococcus pneumoniae topoisomerase III, and thelike, including other type IA topoisomerases (see Berger, Biochim.Biophys. Acta 1400:3-18, 1998; DiGate and Marians, J. Biol.Chem._264:17924-17930, 1989; Kim and Wang, J. Biol. Chem.267:17178-17185, 1992; Wilson et al., J. Biol. Chem. 275:1533-1540,2000; Hanai et al., Proc. Natl. Acad. Sci., USA 93:3653-3657, 1996, U.S.Pat. No. 6,277,620, each of which is incorporated herein by reference).E. coli topoisomerase 111, which is a type IA topoisomerase thatrecognizes, binds to and cleaves the sequence 5′-GCAACTT-3′, can beparticularly useful in a method of the invention (Zhang et al., J. Biol.Chem. 270:23700-23705, 1995, which is incorporated herein by reference).A homolog, the traE protein of plasmid RP4, has been described by Li etal. (J. Biol. Chem. 272:19582-19587, 1997) and can also be used in thepractice of the invention. A DNA-protein adduct is formed with theenzyme covalently binding to the 5′-thymidine residue, with cleavageoccurring between the two thymidine residues.

Type IB topoisomerases include the nuclear type I topoisomerases presentin all eukaryotic cells and those encoded by vaccinia and other cellularpoxviruses (see Cheng et al., Cell 92:841-850, 1998, which isincorporated herein by reference). The eukaryotic type IB topoisomerasesare exemplified by those expressed in yeast, Drosophila and mammaliancells, including human cells (see Caron and Wang, Adv. Pharmacol.29B,:271-297, 1994; Gupta et al., Biochim. Biophys. Acta 1262:1-14,1995, each of which is incorporated herein by reference; see, also,Berger, supra, 1998). Viral type IB topoisomerases are exemplified bythose produced by the vertebrate poxviruses (vaccinia, Shope fibromavirus, ORF virus, fowlpox virus, and molluscum contagiosum virus), andthe insect poxvirus (Amsacta moorei entomopoxvirus) (see Shuman,Biochim. Biophys. Acta 1400:321-337, 1998; Petersen et al., Virology230:197-206, 1997; Shuman and Prescott, Proc. Natl. Acad. Sci., USA84:7478-7482, 1987; Shuman, J. Biol. Chem. 269:32678-32684, 1994; U.S.Pat. No. 5,766,891; PCT/US95/16099; PCT/US98/12372, each of which isincorporated herein by reference; see, also, Cheng et al., supra, 1998).

Type II topoisomerases include, for example, bacterial gyrase, bacterialDNA topoisomerase IV, eukaryotic DNA topoisomerase II, and T-even phageencoded DNA topoisomerases (Roca and Wang, Cell 71:833-840, 1992; Wang,J. Biol. Chem. 266:6659-6662, 1991, each of which is incorporated hereinby reference; Berger, supra, 1998). Like the type IB topoisomerases, thetype II topoisomerases have both cleaving and ligating activities. Inaddition, like type IB topoisomerase, substrate ds nucleotide sequencescan be prepared such that the type II topoisomerase can form a covalentlinkage to one strand at a cleavage site. For example, calf thymus typeII topoisomerase can cleave a substrate ds nucleotide sequencecontaining a 5′ recessed topoisomerase recognition site positioned threenucleotides from the 5′ end, resulting in dissociation of the threenucleotide sequence 5′ to the cleavage site and covalent binding the ofthe topoisomerase to the 5′ terminus of the ds nucleotide sequence(Andersen et al., supra, 1991). Furthermore, upon contacting such a typeII topoisomerase-charged ds nucleotide sequence with a second nucleotidesequence containing a 3′ hydroxyl group, the type II topoisomerase canligate the sequences together, and then is released from the recombinantnucleic acid molecule. As such, type II topoisomerases also are usefulfor performing methods of the invention.

Structural analysis of topoisomerases indicates that the members of eachparticular topoisomerase families, including type IA, type IB and typeII topoisomerases, share common structural features with other membersof the family (Berger, supra, 1998). In addition, sequence analysis ofvarious type IB topoisomerases indicates that the structures are highlyconserved, particularly in the catalytic domain (Shuman, supra, 1998;Cheng et al., supra, 1998; Petersen et al., supra, 1997). For example, adomain comprising amino acids 81 to 314 of the 314 amino acid vacciniatopoisomerase shares substantial homology with other type IBtopoisomerases, and the isolated domain has essentially the sameactivity as the full length topoisomerase, although the isolated domainhas a slower turnover rate and lower binding affinity to the recognitionsite (see Shuman, supra, 1998; Cheng et al., supra, 1998). In addition,a mutant vaccinia topoisomerase, which is mutated in the amino terminaldomain (at amino acid residues 70 and 72) displays identical propertiesas the full length topoisomerase (Cheng et al., supra, 1998). In fact,mutation analysis of vaccinia type IB topoisomerase reveals a largenumber of amino acid residues that can be mutated without affecting theactivity of the topoisomerase, and has identified several amino acidsthat are required for activity (Shuman, supra, 1998). In view of thehigh homology shared among the vaccinia topoisomerase catalytic domainand the other type IB topoisomerases, and the detailed mutation analysisof vaccinia topoisomerase, it will be recognized that isolated catalyticdomains of the type IB topoisomerases and type IB topoisomerases havingvarious amino acid mutations can be used in the methods of theinvention.

The various topoisomerases exhibit a range of sequence specificity. Forexample, type II topoisomerases can bind to a variety of sequences, butcleave at a highly specific recognition site (see Andersen et al., J.Biol. Chem. 266:9203-9210, 1991, which is incorporated herein byreference). In comparison, the type IB topoisomerases include sitespecific topoisomerases, which bind to and cleave a specific nucleotidesequence (“topoisomerase recognition site”). Upon cleavage of a dsnucleotide sequence by a topoisomerase, for example, a type IBtopoisomerase, the energy of the phosphodiester bond is conserved viathe formation of a phosphotyrosyl linkage between a specific tyrosineresidue in the topoisomerase and the 3′ nucleotide of the topoisomeraserecognition site. Where the topoisomerase cleavage site is near the 3′terminus of the nucleic acid molecule, the downstream sequence (3′ tothe cleavage site) can dissociate, leaving a nucleic acid moleculehaving the topoisomerase covalently bound to the newly generated 3′ end(see FIG. 1).

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, can be performed bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein the first ds nucleotide sequence has a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) at or near the 5′ terminus of the firstend or the second end or both; 2) at least a second ds nucleotidesequence that has, or can be made to have, a first end and a second end;and 3) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)site-specific topoisomerase (e.g., a type IA or a type IB topoisomeraserecognition site), under conditions such that all components are incontact and the at least one topoisomerase can effect its activity. Forexample, the topoisomerase can be a type IA topoisomerase such as E.coli topoisomerase I, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a ds nucleotide sequence, thetopoisomerase preferably is stably bound to the 5′ terminus. Preferably,upon cleavage by the topoisomerase, the cleaved ds nucleotide sequencecomprises a 3′ overhanging sequence.

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand can be performed such that anycombination of ends are linked, and wherein one strand at the ends beinglinked is covalently linked and the other strand is not covalentlylinked, but contains a nick. For example, the first ds nucleotidesequence can comprise a coding sequence, wherein the ATG start codon isat or near the first end and a poly A signal is encoded at or near thesecond end; and a second ds nucleotide sequence can comprise a promoterelement, which functions when positioned upstream of a coding sequence,and the first end is upstream of the second end, the method can beperformed wherein a site-specific topoisomerase recognition site (e.g.,a type IA or a type II topoisomerase recognition site) is at or near the5′ terminus of the first end of the first ds nucleotide sequence, andwherein the contacting is performed under conditions such that thetopoisomerase (e.g., a type IA or a type II topoisomerase) cancovalently link the 5′ terminus of the first end of the first dsnucleotide sequence to the 3′ terminus of the first end of the second dsnucleotide sequence, thereby generating a ds recombinant nucleic acidmolecule, in which a polypeptide can be expressed from the codingsequence. Alternatively, the method can be performed wherein thetopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of thesecond end of the first ds nucleotide sequence, and wherein thecontacting is performed under conditions such that the topoisomerase(e.g., a type IA or a type II topoisomerase recognition site) cancovalently link the 5′ terminus of the second end of the first dsnucleotide sequence to the 3′ terminus of the first end of the second dsnucleotide sequence, thereby generating a ds recombinant nucleic acidmolecule from which an antisense molecule can be expressed.

As another example using the first ds nucleotide sequence and second dsnucleotide sequence described above, the method can be performed,wherein the topoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of each ofthe first end and the second end of the first ds nucleotide sequence,and wherein the contacting is performed under conditions such that thetype IA topoisomerase can covalently link the 5′ terminus of the firstend of the first ds nucleotide sequence to the 3′ terminus of the firstend of the second ds nucleotide sequence, and the 5′ terminus of thesecond end of the first ds nucleotide sequence to the 3′ terminus of thesecond end of the second ds nucleotide sequence. As such, the dsrecombinant nucleic acid molecule generated by the method iscircularized, and includes a nick in each strand opposite the locationwhere a strand was covalently linked by a topoisomerase (e.g., a type IAor a type II topoisomerase). Furthermore, the promoter of the second dsnucleotide sequence can initiate expression of the first ds nucleotidesequence. In one embodiment, the circularized ds recombinant nucleicacid molecule comprises a vector.

As another example using the first ds nucleotide sequence and second dsnucleotide sequence described above, the method can be performed,wherein the topoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of each ofthe first end and the second end of the first ds nucleotide sequence,and wherein the contacting is performed under conditions such that thetopoisomerase (e.g., a type IA or a type II topoisomerase) cancovalently link the 5′ terminus of the first end of the first dsnucleotide sequence to the 3′ terminus of the second end of the secondds nucleotide sequence, and the 5′ terminus of the second end of thefirst ds nucleotide sequence to the 3′ terminus of the first end of thesecond ds nucleotide sequence. As such, the ds recombinant nucleic acidmolecule generated by the method is circularized, and includes a nick ineach strand opposite the location where a strand was covalently linkedby topoisomerase (e.g., a type IA or a type II topoisomerase recognitionsite). Furthermore, the promoter of the second ds nucleotide sequencecan initiate expression of an antisense sequence. In one embodiment, thecircularized ds recombinant nucleic acid molecule comprises a vector.

As disclosed herein, a method of generating a ds recombinant nucleicacid molecule covalently linked in one strand, involving a first dsnucleotide sequence and at least a second ds nucleotide sequence, canfurther include a step for amplifying the ds recombinant nucleic acidmolecule covalently linked in one strand. The amplification reaction canbe carried out by contacting the ds recombinant nucleic acid moleculewith an amplification reaction primer pair, wherein a first primer ofthe pair is capable of binding to the covalently linked strand, at ornear one end of the first or second ds nucleotide sequence, and primingan amplification reaction toward the other ds nucleotide sequence togenerate a first extension product that is identical in nucleotidesequence to the nicked strand of the ds recombinant nucleic acidmolecule; and the second primer of the pair is capable of binding to thefirst extension product, typically at or near the 3′ terminus, and, inthe presence of the first primer, can generate an amplification productusing the covalently linked strand and the extension product (orextension products generated therefrom) as templates. For example, themethod can be performed such that the type IA topoisomerase recognitionsite is at or near a first end of the first ds nucleotide sequence, andthe method further includes contacting the ds recombinant nucleic acidmolecule with an amplification reaction primer pair, wherein a forwardprimer is capable of binding at or near the second end of the first dsnucleotide sequence, and wherein a reverse primer is capable of bindingto a nucleotide sequence complementary to at least a portion of thesecond end of the second ds nucleotide sequence; and amplifying the dsrecombinant nucleic acid molecule. The first ds nucleotide sequence caninclude a coding region and the second ds nucleotide sequence caninclude a regulatory element.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in one strand also can be performed by contacting 1) a first dsnucleotide sequence having a first end and a second end, wherein thefirst ds nucleotide sequence has a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite) at or near the 5′ terminus of the first end or the second end orboth; 2) at least a second ds nucleotide sequence that has, or can bemade to have, a first end and a second end; 3) at least a third dsnucleotide sequence which has, or can be made to have, a first end and asecond end, each end further comprising a 5′ terminus and a 3′ terminus;and 4) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)site-specific topoisomerase (e.g., a type IA or a type II topoisomeraserecognition site), under conditions such that all components are incontact and the at least one topoisomerase can effect its activity. Forexample, the topoisomerase can be a type IA topoisomerase such as E.coli topoisomerase I, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a ds nucleotide sequence, thetopoisomerase preferably is stably bound to the 5′ terminus. Preferably,upon cleavage by the topoisomerase, the cleaved ds nucleotide sequencecomprises a 3′ overhanging sequence.

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, involving a first dsnucleotide sequence that contains a site-specific topoisomeraserecognition site (e.g., a type IA or a type IB topoisomerase recognitionsite), or cleavage product thereof, at least a second ds nucleotidesequence, and at least a third ds nucleotide sequence can be performedsuch that any combination of ends are linked, and one strand at the endsbeing linked is covalently linked and one strand is nicked. According tothis embodiment, any of the ends can contain a type IA, type II, or typeIB topoisomerase recognition site, or can comprise a cleavage productthereof, provided that the first ds recombinant nucleotide moleculecontains a topoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) at or near a 5′ terminus, or a cleavageproduct thereof, and only one topoisomerase or topoisomerase recognitionsite is present at the ends that are to be linked. For example, wherethe first ds nucleotide sequence comprises a site-specific type IAtopoisomerase recognition site at or near each of the first end and thesecond end, the method further can include contacting the first dsnucleotide sequence and the second ds nucleotide sequence with at leasta third ds nucleotide sequence which has, or can be made to have, afirst end and a second end, each end further comprising a 5′ terminusand a 3′ terminus, under conditions such that the topoisomerase (e.g., atype IA or a type II topoisomerase) can covalently link the 5′ terminusof the first end of the first ds nucleotide sequence with the 3′terminus of the first end of the second nucleotide sequence, and the 5′terminus of the second end of the first ds nucleotide sequence with the3′ terminus of the first end of the third nucleotide sequence. It willbe recognized that other combinations of ends and topoisomeraserecognition sites, or cleavage products thereof, can be used to performsuch a method of the invention.

A method of the invention also can be performed by contacting a first dsnucleotide sequence and a second ds nucleotide sequence with at least athird ds nucleotide sequence, which comprises a first end and a secondend, each end further comprising a 5′ terminus and a 3′ terminus,wherein the third ds nucleotide sequence comprises a type IBtopoisomerase recognition site at or near the 3′ terminus of said firstend, or said second end, or both said first end and said second end; andat least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) type IBtopoisomerase under conditions such that the type IB topoisomerase cancovalently link the 3′ terminus of the first end or second end of thethird ds nucleotide sequence to the 5′ terminus of the first end orsecond end of the second ds nucleotide sequence. In such a method, wherethe third ds nucleotide sequence comprises a type IB topoisomeraserecognition site at or near the 3′ terminus of the first end, thecontacting can be performed under conditions such that the type IBtopoisomerase can covalently link the 3′ terminus of the first end ofthe third ds nucleotide sequence to the 5′ terminus of the first end ofthe second ds nucleotide sequence. It will be recognized that othercombinations of ends and topoisomerase recognition sites, or cleavageproducts thereof, can be used to perform such a method of the invention.

In another embodiment, a method for generating a ds recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting 1) a first ds nucleotide sequence having a first end and asecond end, wherein the first ds nucleotide sequence has a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) at or near the 5′ terminus of an end anda type IB topoisomerase recognition site at or near the 3′ terminus ofthe other end; 2) at least a second ds nucleotide sequence that has, orcan be made to have, a first end and a second end; 3) at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site-specific topoisomerase(e.g., a type IA or a type II topoisomerase); and 4) at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) type IB topoisomerase underconditions such that all components are in contact and the at least onetopoisomerase can effect its activity. For example, the topoisomerase,for which a recognition site is at or near the 5′ terminus, can be atype IA topoisomerase such as E. coli topoisomerase 1, E. colitopoisomerase III, or a eukaryotic topoisomerase III. Upon cleavage of ads nucleotide sequence, the type IA topoisomerase preferably is stablybound to the 5′ terminus, and the type IB topoisomerase preferably isstably bound at the 3′ terminus. Preferably, upon cleavage by thetopoisomerases, the cleaved ds nucleotide sequence comprises a 3′overhanging sequence and a 5′ overhanging sequence. The method canfurther include contacting the ds recombinant nucleic acid molecule witha DNA ligase, thereby generating a ds recombinant nucleic acid moleculecovalently linked in both strands.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in one strand by contacting a first ds nucleotide sequence, asecond ds nucleotide sequence, and at least a third ds nucleotidesequence, can further include a step for amplifying the ds recombinantnucleic acid molecule, particularly the covalently linked strand. Theamplification can be carried out by contacting the ds recombinantnucleic acid molecule with an amplification reaction primer pair,wherein a first primer of the pair can bind selectively to thecovalently linked strand at or near one end of the first or second dsnucleotide sequence and prime an amplification reaction toward the otherds nucleotide sequence to generate a first extension product that iscomplementary to the covalently-linked strand; and the second primer ofthe pair can bind selectively to the first extension product, typicallyat or near the 3′ terminus, and, in the presence of the first primer,can generate an amplification product using the covalently linked strandand the extension product (or extension products derived therefrom) astemplates. The method can be performed such that the topoisomeraserecognition site (e.g., a type IA or a type IB topoisomerase recognitionsite) is at or near the first end of the first ds nucleotide sequence,and can further include contacting the ds recombinant nucleic acidmolecule with an amplification reaction primer pair, wherein a forwardprimer is capable of binding to a nucleotide sequence at or near thesecond end of the first ds nucleotide sequence and wherein a reverseprimer is capable of binding to a nucleotide sequence complementary toat least a portion of the third ds nucleotide sequence; and amplifyingthe ds recombinant nucleic acid molecule. The first ds nucleotidesequence can include a coding region and the third ds nucleotidesequence can include a regulatory element. Furthermore, the ends beinglinked can contain complementary overhanging sequences.

Representative embodiments of the disclosed methods for generating a dsrecombinant nucleic acid molecule covalently linked in one strand areillustrated in FIGS. 4A-4F. In FIG. 4A, one of the ds nucleotidesequences has a topoisomerase attached to the 5′ terminus of one endsuch that, when this molecule, which has a 3′ overhang, is contactedwith a second ds nucleotide sequence having a substantiallycomplementary 3′ overhang, under suitable conditions, the nucleotidescomprising the 3′ overhangs can hybridize and the topoisomerases cancatalyze ligation. FIG. 4B shows a first ds nucleotide sequence havingtopoisomerase molecules linked to the 5′ terminus and 3′ terminus of twodifferent ends of one nucleotide sequence, and further shows linkage ofthe first ds nucleotide sequence to two other nucleotide sequences togenerate a nucleic acid molecule which has one strand without any nicksand another strand with two nicks. FIG. 4C shows a first ds nucleotidesequence having a topoisomerase molecule linked to the 5′ terminus ofone end and a second ds nucleotide sequence having a topoisomerasemolecule linked to the 5′ terminus of one end, and further shows linkageof the first and second ds nucleotide sequence to one other nucleotidesequence to generate a nucleic acid molecule which has one strandwithout any nicks and another strand with two nicks. In FIG. 4D, one ofthe ds nucleotide sequences to be linked has site-specific type IAtopoisomerases attached to the 5′ terminus of both ends such that, whenthe nucleotide sequences are contacted the complementary 3′ overhangscan hybridize and the topoisomerases catalyze ligation. FIG. 4E showsanother example of linking three ds nucleotide sequences together, usingone ds nucleotide sequence that is topoisomerase-charged with a type IAtopoisomerase at a 5′ terminus and another ds nucleotide sequence thatis topoisomerase-charged with a type IB topoisomerase at a 3′ terminusof the opposite strand to be linked, such that when the nucleotidesequences are contacted the complementary 3′ overhangs can hybridize andthe topoisomerases catalyze ligation. FIG. 4F illustrates anotherexample of linking three ds nucleotide sequences together, in this caseusing one ds nucleotide sequence that is topoisomerase-charged with atopoisomerase (e.g., a type IA or a type II topoisomerase) at a 5′terminus and with a type IB topoisomerase at a 3′ terminus of theopposite strand, such that when the nucleotide sequences are contactedunder suitable conditions, the complementary 3′ overhangs can hybridizeand the topoisomerases catalyze ligation.

The examples set forth in FIGS. 4A-4F show the ends of the ds nucleotidesequences opposite those being linked as having blunt ends, and showsthe being linked as having 3′ overhanging sequences. However, thesubstrate ds nucleotide sequences can have any ends and overhangs asdesired, including both ends being blunt and/or complementary, orcombinations thereof, such that the ends can be ligated to each other,for example, to form circular molecules or to other nucleic acidmolecules having an appropriate end. Thus, one or more of the blunt endsas shown in FIGS. 4A-4F can be substituted with a nucleotide sequencecomprising a 5′ overhang or a 3′ overhang, either of which canconstitute a single nucleotide such as a thymidine residue or multiplenucleotides (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, etc. nucleotides), whichcan be the same or different. In certain embodiments of the disclosedmethods, a first ds nucleotide sequence contains a blunt end to belinked, and a second ds nucleotide sequence contains an overhang at theend which is to be linked by a site-specific topoisomerase (e.g., a typeIA or a type IB topoisomerase), wherein the overhang includes a sequencecomplementary to that comprising the blunt end, thereby facilitatingstrand invasion as a means to properly position the ends for the linkingreaction.

As exemplified in FIGS. 4A-4C, the ds recombinant nucleic acid moleculegenerated using the methods of this aspect of the invention includethose in which one strand (not both strands) is covalently linked at theends to be linked (i.e. ds recombinant nucleic acid molecules generatedusing these methods contain a nick at each position where two ends werejoined). These embodiments are particularly advantageous in that apolymerase can be used to replicate the ds recombinant nucleic acidmolecule by initially replicating the covalently linked strand. Forexample, a thermostable polymerase such as a polymerase useful forperforming an amplification reaction such as PCR can be used toreplicate the covalently strand, whereas the strand containing the nickdoes not provide a suitable template for replication.

The present invention also provides methods of covalently ligating theends of two different ds nucleotide sequences or two ends of the same dsnucleotide sequence, such that the product generated is ligated in bothstrands and, therefore, does not contain a nick. Representativeembodiments of this aspect of the invention are illustrated in FIG. 5.For example, in FIG. 5A, one of the ds nucleotide sequences hastopoisomerase molecules attached to the 3′ terminus and the 5′ terminusof one end such that, when this molecule, which has a 5′ overhang, iscontacted with a second ds nucleotide sequence having a substantiallycomplementary 5′ overhang, under suitable conditions, the nucleotidescomprising the 5′ overhangs can hybridize and the topoisomerases cancatalyze ligation of both strands of the ds nucleotide sequences. InFIG. 5B, each end of the ds nucleotide sequences to be linked has atopoisomerase molecule attached to the 3′ terminus such that, when thenucleotide sequences are contacted under suitable conditions,nucleotides comprising the 5′ overhangs can hybridize and thetopoisomerases catalyze ligation (compare FIG. 5C, in which each of theds nucleotide sequences to be linked has a topoisomerase attached to the5′termini of the ends to be linked). FIG. 5D illustrates linking threeds nucleotide sequences together via a ds nucleotide sequence that istopoisomerase-charged at both termini of both ends. Similarly to FIG. 4,the examples set forth in FIGS. 5A-5D show the ends of the ds nucleotidesequences that are not being linked as having blunt ends. As discussedwith respect to FIG. 4, however, the substrate ds nucleotide sequencesutilized in methods as exemplified in FIG. 5 can have any ends asdesired, including topoisomerase-charged ends, such that the ends can beligated to each other, for example, to form circular molecules or toother nucleic acid molecules having an appropriate end, blunt ends, 5′overhangs, 3′ overhangs, and the like, as desired.

A covalently bound topoisomerase, in addition to catalyzing a ligationreaction, also can catalyze the reverse reaction, for example,religation of the 3′ nucleotide of the recognition sequence, to whichthe type IB topoisomerase is linked through the phosphotyrosyl bond, andthe nucleotide sequence that, prior to cleavage, comprised the 5′terminus of the ds nucleotide sequence, and which, following cleavage,contains a free 5′ hydroxy group. As such, methods have been developedfor using a type IB topoisomerase to produce recombinant nucleic acidmolecules. For example, cloning vectors containing a bound type IBtopoisomerase have been developed and are commercially available(Invitrogen Corp., La Jolla Calif.). Such cloning vectors, whenlinearized, contain a covalently bound type IB topoisomerase at each 3′end (“topoisomerase-charged”). Nucleotide sequences such as thosecomprising a cDNA library, or restriction fragments, or sheared genomicDNA sequences that are to be cloned into such a vector are treated, forexample, with a phosphatase to produce 5′ hydroxyl termini, then areadded to the linearized topoisomerase-charged vector under conditionsthat allow the topoisomerase to ligate the nucleotide sequences at the5′ terminus containing the hydroxyl group and the 3′ terminus of thevector that contains the covalently bound topoisomerase. A nucleotidesequence such as a PCR amplification product, which is generatedcontaining 5′ hydroxyl ends, can be cloned into a topoisomerase-chargedvector in a rapid joining reaction (approximately 5 minutes at roomtemperature). The rapid joining and broad temperature range inherent tothe topoisomerase joining reaction makes the use oftopoisomerase-charged vectors ideal for high throughput applications,which generally are performed using automated systems.

Type II topoisomerases have not generally been used for generatingrecombinant nucleic acid molecules or cloning procedures, whereas typeIB topoisomerases, as indicated above, are used in a variety ofprocedures. As disclosed herein, type IA topoisomerases can be used in avariety of procedures similar to those described for the type IBtopoisomerases. However, previously described methods of using type IBtopoisomerases to ligate two or more nucleotide sequences have sufferedfrom the disadvantage that the bound topoisomerase only effects thejoining of the 3′ end of the strand to which it is attached and a secondstrand containing a 5′ hydroxyl group. Since the topoisomerase cannotligate the complementary strands, the nucleic acid molecules that aregenerated contain nicks. While the presence of such nicks does notprevent the use of the recombinant molecules for transfection of a hostcells, as the nicks generally are resolved intracellularly, the presenceof such nicks in double stranded nucleic acid molecules significantlylimits direct use of the recombinant molecules. For example, a strand ofa nucleic acid molecule containing a nick cannot be amplified by PCRbecause the primer extension reaction terminates at the nick. Thus,nucleic acid constructs prepared using a topoisomerase according topreviously described methods generally must be further treated, forexample, with a DNA ligase, to obtain a ds recombinant nucleic acidmolecule that is covalently linked in both strands and, therefore,useful for subsequent manipulations such as PCR.

Previously described methods for preparing nucleic acid constructs alsogenerally required numerous steps, particularly where more than twonucleotide sequences are to be ligated, and even more so where thesequences must be ligated in a predetermined orientation. For example,the nucleotide sequences to be linked generally are ligated sequentiallyto produce intermediate constructs, each of which must be cloned,amplified in a host cell, isolated, and characterized. The constructscontaining the correct sequences then must be isolated in a sufficientquantity and form such that the next nucleotide sequence can be ligated,and the process of cloning, amplifying, isolating and characterizingperformed again to identify the proper construct. Clearly, as the numberof different nucleotide sequences to be joined increases, so do thenumber of essentially repetitive procedures that must be performed, thusresulting in an expensive, laborious and lengthy process.

As disclosed herein, an advantage of a method of the invention forgenerating a ds recombinant nucleic acid molecule covalently linked inboth strands is that there is no need to perform a separate ligationreaction in order to obtain a functional ds recombinant nucleic acidmolecule covalently linked in both strands (see FIGS. 1 and 5). Inaddition, a method of this aspect of the invention can be performed suchthat, where a number of different ds nucleotide sequences are to becovalently linked in a predetermined orientation, there is norequirement that intermediate constructs be cloned, characterized andisolated before proceeding to a subsequent step (see Example 1.B). Assuch, the methods of this aspect of the invention provide a means togenerate a ds recombinant nucleic acid molecule covalently linked inboth strands much more quickly and at a substantially lower cost thanwas possible using previously known methods.

As an additional advantage, the generated ds recombinant nucleic acidmolecules covalently linked in both strands are in a form that can beused directly in further procedures, for example, particular proceduresinvolving extension or a primer such as a PCR amplification procedure,or other transcription or translation procedure, because the generatedconstruct does not contain nicks at the sites where the ds nucleotidessequences have been joined. As disclosed herein, a method of theinvention for generating a ds recombinant nucleic acid moleculecovalently linked in one strand, in certain embodiments, also isadvantageous in that the generated ds recombinant nucleic acid moleculesare in a form that can be used directly in further procedures, forexample, particular procedures involving extension of a primer such as aPCR amplification procedure, or other transcription or translationprocedure, because in certain embodiments, the generated ds recombinantnucleic acid molecule contains one strand that does not contain a nickat the sites where the ds nucleotides sequences were joined.

The term “nucleotide sequence” or “ds nucleotide sequence” is usedherein to refer to a discrete nucleic acid molecule. When used as such,the term “nucleotide sequence” is used merely for convenience such thatthe components in a composition or used in a method of the invention canbe clearly distinguished. Thus, reference is made, for example, to “dsnucleotide sequences”, which, in a method of the invention, correspondto the reactants (substrates) used to produce a recombinant “nucleicacid molecule” product.

Certain methods of the invention are exemplified generally herein withreference to the use of type IB topoisomerase such as the Vacciniatopoisomerase, or a type IA topoisomerase. However, it will berecognized that the methods also can be performed using a topoisomeraseother than that exemplified, merely by adjusting the componentsaccordingly. For example, as described in greater detail below, methodsare disclosed for incorporating a type IB topoisomerase recognition siteat one or both 3′ termini of a linear ds nucleotide sequence using a PCRprimer comprising, at least in part, a nucleotide sequence complementaryto the topoisomerase recognition site. In comparison, a topoisomeraserecognition site for a type IA or, if desired, type II topoisomerase,can be incorporated into a ds nucleotide sequence by using a PCR primerthat contains the recognition site.

Cleavage of a ds nucleotide sequence by a site specific type IBtopoisomerase results in the generation of a 5′ overhanging sequence inthe strand complementary to and at the same end as that containing thecovalently bound topoisomerase. Furthermore, as disclosed herein, PCRprimers can be designed that can incorporate a type IB topoisomeraserecognition site into a ds nucleotide sequence, and that further canproduce, upon cleavage of the ds nucleotide sequence by thetopoisomerase, a 5′ overhanging sequence in the complementary strandthat has a defined and predetermined sequence. As such, the methods arereadily adaptable to generating a ds recombinant nucleic acid moleculehaving the component ds nucleotide sequence operatively linked in apredetermined orientation. In view of the present disclosure, it will berecognized that PCR primers also can be designed such that a type IAtopoisomerase recognition site can be introduced into a ds nucleotidesequence, including a library of diverse sequences, and, if desired,such that upon cleavage by a site-specific topoisomerase, generates a 3′overhanging sequence.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in both strands, as disclosed herein, extends the previouslyknown methods by providing a topoisomerase at or near the terminus ofeach ds nucleotide sequence to be covalently linked. For example, withrespect to a type IB topoisomerase, the method provides a topoisomeraserecognition site, or a cleavage product thereof (i.e., a covalentlybound type IB topoisomerase), at or near the 3′ terminus of each lineards nucleotide sequence to be linked. As used herein, the term“topoisomerase recognition site” means a defined nucleotide sequencethat is recognized and bound by a site specific topoisomerase. Forexample, the nucleotide sequence 5′-(C/T)CCTT-3′ is a topoisomeraserecognition site that is bound specifically by most poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I, which thencan cleave the strand after the 3′-most thymidine of the recognitionsite to produce a nucleotide sequence comprising 5′-(C/T)CCTT-PO₄-TOPO,i.e., a complex of the topoisomerase covalently bound to the 3′phosphate through a tyrosine residue in the topoisomerase (see Shuman,J. Biol. Chem. 266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. AcidsRes. 22:5360-5365, 1994; each of which is incorporated herein byreference; see, also, U.S. Pat. No. 5,766,891; PCT/US95/16099;PCT/US98/12372). In comparison, the nucleotide sequence 5′-GCAACTT-3′ isthe topoisomerase recognition site for type IA E. coli topoisomeraseIII.

Topoisomerase-charged ds nucleotide sequences, including thosecontaining a topoisomerase covalently attached to a 5′ terminus or 3′terminus or both, of one or both ends of the ds nucleotide sequence, canbe generated by any of a number of methods. In some cases and under theappropriate conditions, type I topoisomerases can cleave a singlestranded nucleotide sequence. For example, a domain comprising theamino-terminal 67 kDa domain of E. coli topoisomerase I, which is a typeIA topoisomerase, can cleave a single stranded nucleotide sequencecontaining the topoisomerase recognition site. Where conditions are suchthat the topoisomerases can cleave a single stranded nucleotidesequence, cleavage of a ds nucleotide sequence containing topoisomeraserecognition sites at the 5′ and 3′ termini of one end of ds nucleotidesequence can be performed in parallel. Alternatively, where one or bothof the topoisomerases requires a ds nucleotide sequence for recognitionand cleavage, the reactions are performed serially, wherein the moreterminal (distal) of the topoisomerase recognition sites is cleavedfirst, then the more internal (proximal) site, which remains in a doublestranded context, is cleaved. For example, a ds nucleotide sequencecontaining an E. coli topoisomerase III recognition site at or near a 5′terminus of an end and a Vaccinia type IB topoisomerase recognition siteat or near the 3′ terminus of the same end, and wherein the type IBrecognition site is closer to the end than the type IA recognition site,the ds nucleotide sequence can be incubated with the Vacciniatopoisomerase, to produce a type IB topoisomerase-charged ds nucleotidesequence, then with the E. coli topoisomerase, to produce a dsnucleotide sequence having the type IA topoisomerase bound to the 5′terminus and the type IB topoisomerase bound to the 3′ terminus.Accordingly, the invention includes methods for producing ds nucleotidesequence comprising a topoisomerase attached to one or both termini ofat least one end, and further provides such topoisomerase-charged dsnucleotide sequences.

As used herein, the term “cleavage product,” when used in reference to atopoisomerase recognition site, refers to a nucleotide sequence that hasbeen cleaved by a topoisomerase, generally at its recognition site, andcomprises a complex of the topoisomerase covalently bound, in the caseof type IA or type II topoisomerase, to the 5′ phosphate group of the 5′terminal nucleotide in the topoisomerase recognition site, or in thecase of a type IB topoisomerase to the 3′ phosphate group of the 3′terminal nucleotide in the topoisomerase recognition site. Such acomplex, which comprises a topoisomerase cleaved ds nucleotide sequencehaving the topoisomerase covalently bound thereto, is referred to hereinas a “topoisomerase-activated” or a “topoisomerase-charged” nucleotidesequence. Topoisomerase-activated ds nucleotide sequences can be used ina method of the invention, as can ds nucleotide sequences that containan uncleaved topoisomerase recognition site and a topoisomerase, whereinthe topoisomerase can cleave the ds nucleotide sequence at therecognition site and become covalently bound thereto.

In one embodiment of a method of generating a ds recombinant nucleicacid molecule covalently linked in both strands, a topoisomeraserecognition site is present at or near the 3′ terminus of the end ofeach nucleotide sequence to be linked such that, in the presence of atype IB topoisomerase, each nucleotide sequence is cleaved to produce a3′ terminus, which contains the topoisomerase covalently bound thereto(see FIG. 1). The nucleotide sequences to be covalently linked also cancontain a 5′ hydroxy group at the same end as that containing thetopoisomerase recognition site, or a 5′ hydroxyl group can be generatedusing a phosphatase. Upon contact of such nucleotide sequences, the sitespecific topoisomerase can ligate each strand containing a 3′ phosphateto a respective 5′ hydroxyl group, thereby generating a ds recombinantnucleic acid molecule covalently linked in both strands, which can beproduced as a linear, circular, or positively or negatively supercoilednucleic acid molecule.

Preferably, the 5′ termini of the ends of the nucleotide sequences to belinked by a type IB topoisomerase according to a method of certainaspects of the invention contain complementary 5′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 5′ termini of the ends of the nucleotidesequences to be linked by a type IB topoisomerase according to a methodof certain aspects of the invention contain complementary 5′ sequenceswherein one of the sequences contains a 5′ overhanging sequence and theother nucleotide sequence contains a complementary sequence at a bluntend of a 5′ terminus, to facilitate the initial association of thenucleotide sequences through strand invasion, including, if desired, ina predetermined directional orientation. The term “5” “overhang” or “5′overhanging sequence” is used herein to refer to a strand of a dsnucleotide sequence that extends in a 5′ direction beyond the terminusof the complementary strand of the ds nucleotide sequence. Conveniently,a 5′ overhang can be produced as a result of site specific cleavage of ads nucleotide sequence by a type IB topoisomerase (see Example 1).

Preferably, the 3′ termini of the ends of the nucleotide sequences to belinked by a type IA topoisomerase according to a method of certainaspects of the invention contain complementary 3′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 3′ termini of the ends of the nucleotidesequences to be linked by a topoisomerase (e.g., a type IA or a type IItopoisomerase) according to a method of certain aspects of the inventioncontain complementary 3′ sequences wherein one of the sequences containsa 3′ overhanging sequence and the other nucleotide sequence contains acomplementary sequence at a blunt end of a 3′ terminus, to facilitatethe initial association of the nucleotide sequences through strandinvasion, including, if desired, in a predetermined directionalorientation. The term “3 overhang” or “3 overhanging sequence” is usedherein to refer to a strand of a ds nucleotide sequence that extends ina 5′ direction beyond the terminus of the complementary strand of the dsnucleotide sequence. Conveniently, a 3′ overhang can be produced uponcleavage by a type IA or type II topoisomerase.

The 3′ or 5′ overhanging sequences can have any sequence, thoughgenerally the sequences are selected such that they allow ligation of apredetermined end of one ds nucleotide sequence to a predetermined endof a second nucleotide sequence according to a method of the invention(FIG. 2C, see, also Example 1.B). As such, while the 3′ or 5′ overhangscan be palindromic, they generally are not because ds nucleotidesequences having palindromic overhangs can associate with each other,thus reducing the yield of a ds recombinant nucleic acid moleculecovalently linked in both strands comprising two or more ds nucleotidesequences in a predetermined orientation. For example, the 5′overhanging sequences of ds nucleotide sequences shown in FIG. 2A arepalindrome and, therefore, the association, for example, of a first CMVelement with a second CMV element through the AGCT overhang is just aslikely as the association of a CMV element with a GFP element throughthe AGCT overhang. As such, the efficiency of generating a constructcomprising an operatively covalently linked construct containing, inorder from 5′ to 3′, a CMV element, a GFP element and a BGH elementwould be reduced as compared to the efficiency of generating such aconstruct using the elements as shown in FIG. 2C. The elements shown inFIG. 2B contain palindromic overhangs at one end of the GFP element andat the end of the BGH element shown and, therefore, would be lessefficient than the elements of FIG. 2C, but more efficient than those inFIG. 2A, for generating the desired construct.

A nucleotide sequence used in the methods and kits of the currentinvention can be designed to contain a bridging phosphorothioate toprevent religation after topoisomerase-cleavage. For example, where thetopoisomerase is E. coli topoisomerase III, the bridgingphosphorothioate can be incorporated between the two thymidines of theGCAACTT cleavage/recognition sequence. When cleaved, the clippedsequence contains a 3′-SH instead of a 3′-OH, thus preventing religation(see Burgin et al, Nucl. Acids Res. 23:2973-2979, 1995).

A ds nucleotide sequence useful in a method or kit of an aspect of theinvention can be amplified by an amplification method such as PCR tocontain a topoisomerase recognition site at a 3′ or 5′ terminus of anend. Furthermore, one or both primers used for PCR can be designed suchthat, upon cleavage of an amplified ds nucleotide sequence, the cleavedds nucleotide sequence contains a 5′ or 3′ overhang at one or both ends.In one embodiment, PCR primers are designed such that the 5′ overhangingsequence on a first ds nucleotide sequence is complementary to a 5′overhanging sequence on a second (or other) ds nucleotide sequence,thereby facilitating the association of the nucleotide sequences,preferably in a predetermined orientation, whereupon they can becovalently linked according to a method of the invention. In accordancewith the invention, by designing unique overhanging sequences for thedifferent ds nucleotide sequence to be linked, any number of dsnucleotide sequences can be linked in a desired order and/ororientation.

It should be recognized that PCR is used in two ways with respect to themethods of the invention. In one aspect, PCR primers are designed toimpart particular characteristics to a desired ds nucleotide sequence,for example, a ds nucleotide sequence that encodes a transcriptional ortranslational regulatory element or a coding sequence of interest suchas an epitope tag or cell compartmentalization domain. In this aspect,the PCR primers can be designed such that, upon amplification, the dsnucleotide sequence contains a topoisomerase recognition site at one orboth ends, as desired. As disclosed herein, the PCR primer also caninclude an additional sequence such that, upon cleavage of theamplification product by a site specific topoisomerase, the cleaved dsnucleotide sequence contains a 5′ or 3′ overhanging sequence at thetopoisomerase cleaved end. In an embodiment of the invention involving atopoisomerase that binds and cleaves a 5′ terminus (e.g., an embodimentinvolving a type IA topoisomerase), the PCR primers can be designed tocontain a bridging phosphorothioate linkage (see above), which can blockreligation after topoisomerase cleavage and can assist in the generationof a topoisomerase-charged amplification product.

Overhanging sequences generated using PCR can include a singlenucleotide overhang that is generated as an artifact of the PCRreaction. For example, a polymerase such at Taq, which does not have aproof-reading function and has an inherent terminal transferaseactivity, is commonly used, and produces PCR products containing asingle, non-template derived 3′ A overhang at each end. Theseamplification products can be linked to topoisomerase-charged dsnucleotide sequences containing a single 3′ T overhang or a single 3′ dUoverhang, which, for a T/A cloning reaction, can be a vector (see U.S.Pat. Nos. 5,487,993 and 5,856,144, each of which is incorporated hereinby reference), at one or both ends, using the methods of the invention.

PCR also is used to amplify a covalently linked ds recombinant nucleicacid molecule covalently linked in one or both strands, generated by amethod of the invention. For example, as illustrated in FIG. 6, a methodof the invention can generate an expressible ds recombinant nucleic acidmolecule from three substrate ds nucleotide sequences, including anucleotide sequence comprising a promoter, a nucleotide sequencecomprising a coding sequence, and a nucleotide sequence comprising apolyadenylation signal. The generation of the ds recombinant nucleicacid molecule can be facilitated by the incorporation of complementary3′ (or 5′) overhanging sequences at the ends of the ds nucleotidessequences to be joined. For example, the expressible ds recombinantnucleic acid molecule can be generated by contacting a first dsnucleotide sequence having a type IA topoisomerase at a 5′ terminus of afirst end and a type IB topoisomerase at a 3′ terminus of a second endwith a second ds nucleotide sequence and a third double strandednucleotide sequence. By designing a PCR primer pair containing a firstprimer that is specific for a portion of the nucleotide sequencecomprising the promoter that is upstream from the promoter, and a secondprimer that is specific for a portion of the nucleotide sequencecomprising the polyadenylation signal that is down stream of the signal,only a full length functional ds recombinant nucleic molecule containingthe promoter, coding sequence and polyadenylation signal in the correct(predetermined) orientation will be amplified. In particular, partialreaction products, for example, containing only a promoter linked to thecoding sequence, and reaction products containing nicks are notamplified. Thus, PCR can be used to specifically design a ds nucleotidesequence such that it is useful in a method of the invention, and toselectively amplify only those reaction products having the desiredcomponents and characteristics.

As used herein, the term “covalently linked,” when used in reference toa ds recombinant nucleic acid molecule, means that the nucleic acidmolecule is generated from at least two ds nucleotide sequences that areligated together, in both strands, by a topoisomerase mediated ligation.It should be recognized, for example, that a topoisomerase covalentlybound to one of the ds nucleotide sequences to be covalently linked canbe the same as or different from the topoisomerase covalently bound tothe other ds nucleotide sequence. Thus, a Vaccinia topoisomerase can becovalently bound to one ds nucleotide sequence and another poxvirus oreukaryotic nuclear type IB topoisomerase can be bound to the otherstrand. Generally, however, the topoisomerases, where different, aremembers of the same family, for example, type IA or type IB or type II,although, where the topoisomerases are covalently bound, for example, toa 5′ phosphate and generate complementary 3′ overhangs, thetopoisomerase can be from different families, for example, type IA andtype II.

The term “covalently linked” also is used herein in reference to asingle stranded or double stranded nucleic acid molecule that isgenerated from at least two nucleotide sequences that are ligatedtogether in one strand. For example, a ds recombinant nucleic acidmolecule that is generated when a first topoisomerase-charged dsnucleotide sequence that includes one topoisomerase bound at or near a5′ terminus contacts a second ds nucleotide sequence under conditionssuch that the topoisomerases can covalently link the 5′ terminus of thefirst ds nucleotide sequence to which it is bound, to the 3′ terminus ofthe second ds nucleotide sequence, can generate a ds recombinant nucleicacid molecule covalently linked in one strand.

In one embodiment, a ds recombinant nucleic acid molecule covalentlylinked in both strands generated according to a method of the inventiondoes not contain a nick in either strand at the site where twonucleotide sequences are ligated, although it can contain nickselsewhere in the molecule. In a method for generating a ds recombinantnucleic acid molecule covalently linked in one strand, a ds recombinantnucleic acid molecule is generated that contains a nick at least at theposition where ends were linked in the complementary strands. Thisnicked ds recombinant nucleic acid molecule can be converted to a dsrecombinant nucleic acid molecule covalently linked in both strands byintroducing the nicked ds recombinant nucleic acid molecule into a cell,or by subjecting the ds recombinant nucleic acid molecule to a ligationreaction, such as using a ligase, as is well known in the art.

The term “recombinant” is used herein to refer to a nucleic acidmolecule that is produced by linking at least two nucleotide sequencesaccording to a method of the invention. As such, a ds recombinantnucleic acid molecule encompassed within the present invention isdistinguishable from a nucleic acid molecule that may be produced innature, for example, during meiosis. For example, a ds recombinantnucleic acid molecule covalently linked in both strands generatedaccording to a method of certain aspects of the invention can beidentified by the presence of the two topoisomerase recognition sites,one present in each of the complementary strands, at or near the site atwhich the ds nucleotide sequences were joined.

A method of the invention can be performed by contacting a first dsnucleotide sequence having a first end and a second end, wherein at thefirst end or second end or both, the first ds nucleotide sequence has atopoisomerase recognition site, or cleavage product thereof, at or nearthe 3′ terminus and has (or can be made to have, for example, by contactwith a phosphatase) a hydroxyl group at the 5′ terminus of the same end;at least a second ds nucleotide sequence having a first end and a secondend, wherein at the first end or second end or both, the at least secondds nucleotide sequence has a topoisomerase recognition site, or cleavageproduct thereof, at or near the 3′ terminus and has (or can be made tohave) a hydroxyl group at the 5′ terminus of the same end; and atopoisomerase, under conditions such that the components are in contactand the topoisomerase can effect its activity. Upon contact of thetopoisomerase with the first and second (or other) ds nucleotidesequences, and cleavage, where necessary, each nucleotide sequencecomprises at the cleavage site a covalently bound topoisomerase at the3′ terminus and has, or can have, a hydroxyl group at the 5′ terminussuch that, upon contact, the first and at least second nucleotidesequences are covalently linked in both strands. Accordingly, theinvention provides a ds recombinant nucleic acid molecule covalentlylinked in both strands produced by such a method.

As used herein, the term “at or near,” when used in reference to theproximity of a topoisomerase recognition site to the 3′ (type IB) or 5′(type IA or type II) terminus of a nucleotide sequence, means that thesite is within about 1 to 100 nucleotides from the 3′ terminus or 5′terminus, respectively, generally within about 1 to 20 nucleotides fromthe terminus, and particularly within about 2 to 12 nucleotides from therespective terminus. An advantage of positioning the topoisomeraserecognition site within about 10 to 15 nucleotides of a terminus isthat, upon cleavage by the topoisomerase, the portion of the sequencedownstream of the cleavage site can spontaneously dissociate from theremaining nucleotide sequence, which contains the covalently boundtopoisomerase (referred to generally as “suicide cleavage”; see, forexample, Shuman, supra, 1991; Andersen et al., supra, 1991). Where atopoisomerase recognition site is greater than about 12 to 15nucleotides from the terminus, the nucleotide sequence upstream ordownstream of the cleavage site can be induced to dissociate from theremainder of the sequence by modifying the reaction conditions, forexample, by providing an incubation step at a temperature above themelting temperature of the portion of the duplex including thetopoisomerase cleavage site.

An additional advantage of constructing a first or second (or other) dsnucleotide sequence to comprise, for example, a type IB topoisomeraserecognition site about 2 to 15 nucleotides from one or both ends is thata 5′ overhang is generated following cleavage of the ds nucleotidesequence by a site specific topoisomerase. Such a 5′ overhangingsequence, which would contain 2 to 15 nucleotides, respectively, can bedesigned using a PCR method as disclosed herein to have any sequence asdesired. Thus, where a cleaved first ds nucleotide sequence is to becovalently linked to a selected second (or other) ds nucleotide sequenceaccording to a method of the invention, and where the selected sequencehas a 5′ overhanging sequence, the 5′ overhang on the first dsnucleotide sequence can be designed to be complementary to the 5′overhang on the selected second (or other) ds sequence such that the two(or more) sequences are covalently linked in a predetermined orientationdue to the complementarity of the 5′ overhangs. As discussed above,similar methods can be utilized with respect to 3′ overhanging sequencesgenerated upon cleavage by, for example, a type IA or type IItopoisomerase.

As used herein, reference to a nucleotide sequence having “a first end”and “a second end” means that the nucleotide sequence is linear. Asubstrate ds nucleotide sequence can be linear or circular, includingsupercoiled, although, as a result of cleavage by one or moretopoisomerase, a linear topoisomerase-charged ds nucleotide sequencegenerally is produced. For example, a circular ds nucleotide sequencecontaining two type IB topoisomerase recognition sites within about 100nucleotides of each other and in the complementary strands, preferablywithin about twenty nucleotides of each other and in the complementarystrands, can be contacted with a site specific type IB topoisomerasesuch that each strand is cleaved and the intervening sequencedissociates, thereby generating a linear ds nucleotide sequence having atopoisomerase covalently bound to each end.

It should be recognized that reference to a first end or a second end ofa ds nucleotide sequence is not intended to imply any particularorientation of the nucleotide sequence, and is not intended to imply arelative importance of the ends with respect to each other. Where anucleotide sequence having a first end and second end is a doublestranded nucleotide sequence, each end contains a 5′ terminus and a 3′terminus. Thus, reference is made herein, for example, to a nucleotidesequence containing a topoisomerase recognition site at a 3′ terminusand a hydroxyl group at the 5′ terminus of the same end, which can bethe first end or the second end.

A method of the invention can be performed using only a first dsnucleotide sequence and a second ds nucleotide sequence, or canadditionally include a third, fourth or more ds nucleotide sequences asdesired. Generally, each such nucleotide sequence contains atopoisomerase recognition site, or a cleavage product thereof, at ornear at least one 3′ or 5′ terminus, and can contain a hydroxyl group atthe 5′ terminus of the same end, or a hydroxyl group can be generatedusing a phosphatase. Where a nucleotide sequence does not contain atopoisomerase recognition site at or near an end to be linked to asecond nucleotide sequence, a topoisomerase recognition site can beintroduced into the nucleotide sequence using a method as disclosedherein, for example, by PCR amplification of the sequence using a primercomprising a complement of the topoisomerase recognition site.

The terms “first nucleotide sequence,” “second nucleotide sequence,”“third nucleotide sequence,” and the like, are used herein only toprovide a means to indicate which of several nucleotide sequences isbeing referred to. Thus, absent any specifically defined characteristicwith respect to a particular nucleotide sequence, the terms “first,”“second,” “third” and the like, when used in reference to a nucleotidesequence, or a population or plurality of nucleotide sequences, are notintended to indicate any particular order, importance or otherinformation about the nucleotide sequence. Thus, where an exemplifiedmethod refers, for example, to using PCR to amplify a first dsnucleotide sequence such that the amplification product contains atopoisomerase recognition site at one or both ends, it will berecognized that, similarly, a second (or other) ds nucleotide sequencealso can be so amplified.

The term “at least a second nucleotide sequence” is used herein to meanone or more nucleotide sequences in addition to a first nucleotidesequence. Thus, the term can refer to only a second nucleotide sequence,or to a second nucleotide sequence and a third nucleotide sequence (ormore). As such, the term “second (or other) nucleotide sequence” orsecond (and other) nucleotide sequences” is used herein in recognitionof the fact that the term “at least a second nucleotide sequence” canrefer to a second, third or more nucleotide sequences. It should berecognized that, unless indicated otherwise, a nucleotide sequenceencompassed within the meaning of the term “at least a second nucleotidesequence” can be the same or substantially the same as a firstnucleotide sequence. For example, a first and second ds nucleotidesequence can be the same except for having complementary 5′ overhangingsequences produced upon cleavage by a topoisomerase such that the firstand second ds nucleotide sequences can be covalently linked using amethod of the invention. As such, a method of the invention can be usedto produce a concatenate of first and second ds nucleotide sequences,which, optionally, can be interspersed, for example, by a third dsnucleotide sequence such as a regulatory element, and can contain thecovalently linked sequences in a predetermined directional orientation,for example, each in a 5′ to 3′ orientation with respect to each other.

As disclosed herein, a method of the invention provides a means tocovalently link, two or more ds nucleotides in a predetermineddirectional orientation. The term “directional orientation” or“predetermined directional orientation” or “predetermined orientation”is used herein to refer to the covalent linkage, of two or morenucleotide sequences in a particular order. Thus, a method of theinvention provides a means, for example, to covalently link, a promoterregulatory element upstream of a coding sequence, and to covalently linka polyadenylation signal downstream of the coding region to generate afunctional expressible ds recombinant nucleic acid molecule; or tocovalently link two coding sequences such that they can be transcribedand translated in frame to produce a fusion polypeptide.

A method of the invention also can be performed by contacting a first dsnucleotide sequence having a first end and a second end, wherein at thefirst end or second end or both, the first ds nucleotide sequence has atype IB topoisomerase covalently bound at the 3′ terminus(topoisomerase-charged) and has (or can be made to have) a hydroxylgroup at the 5′ terminus of the same end; and at least a second type IBtopoisomerase-charged ds nucleotide sequence, which has (or can be madeto have) a hydroxyl group at the 5′ terminus at the same end. Uponcontact of the topoisomerase-activated first and at least secondnucleotide sequences at the ends containing the topoisomerase and a 5′hydroxyl group, phosphodiester bonds are formed in each strand, therebygenerating a ds recombinant nucleic acid molecule covalently linked inboth strands.

The invention further provides methods for linking two or more (e.g.,two, three, four, five, six, seven, etc.) nucleotide sequences, whereinthe linked ds recombinant nucleic acid molecule is covalently linked inone strand, but not both strands, (i.e. the ds recombinant nucleic acidmolecule contains a nick in one strand at each position where two endswere joined to generate the ds recombinant nucleic acid molecule. Usingthe schematic shown in FIG. 4A for purposes of illustration, theinvention includes methods for linking at least two nucleotide sequencescomprising contacting a first ds nucleotide sequence having a first endand a second end, wherein at the first end at the second end or at bothends, the first ds nucleotide sequence has a site-specific type IAtopoisomerase covalently bound to the 5′ termini; and a second dsnucleotide sequence which does not have topoisomerase covalently boundto either termini of at least one end. Further, the second nucleotidesequence will typically have hydroxyl groups at the 3′ termini of theend being joined to the first ds nucleotide sequence. In many instances,the two nucleotide sequences to be joined will have either 3′ or 5′overhangs with sufficient sequence complementarity to allow forhybridization. In related embodiments, the first and second dsnucleotide sequences described above may be first and second ends of thesame ds nucleotide sequence. Thus, connection of the two ends results inthe formation of a circularized molecule.

Using the schematic shown in FIG. 4B for purposes of illustration, theinvention includes methods for joining three or more nucleotidesequences. While any number of variations of the invention are possible,three nucleotide sequences may be joined by the use of a linker moleculewhich contains topoisomerases at both the 5′ and 3′ termini of one end.Thus, upon joining of the three nucleotide sequences, a singlenucleotide sequence is formed which contains a first strand with nonicks at the junction points, and a second strand with nicks at thejunction points. This process has the advantage of employing a singletopoisomerase modified molecule to join three nucleotide sequencestogether.

The invention further provides methods for covalently linking bothstrands of two or more (e.g., two, three, four, five, six, seven, etc.)ds nucleotide sequences. Using the schematic shown in FIG. 5A forpurposes of illustration, the invention includes methods for linking atleast two nucleotide sequences comprising contacting a first dsnucleotide sequence having a first end and a second end, wherein at thefirst end at the second end or at both ends, the first ds nucleotidesequence has two topoisomerases (e.g., a type IA and a type IBtopoisomerase) one each covalently bound to the 3′ and 5′ termini; and asecond ds nucleotide sequence which does not have topoisomerasecovalently bound to either termini of at least one end. Further, thesecond nucleotide sequence will often have hydroxyl groups at the 5′ and3′ termini of the end being joined to the first ds nucleotide sequence.In many instances, the two nucleotide sequences to be joined will haveeither 3′ or 5′ overhangs with sufficient sequence complementarity toallow for hybridization. In related embodiments, the first and second dsnucleotide sequences as described above can be first and second ends ofthe same ds nucleotide sequence. Thus, connection of the two endsresults in the formation of a circularized molecule.

Using the schematic shown in FIG. 5D for purposes of illustration, theinvention includes methods for joining three or more nucleotidesequences. While any number of variations of the invention are possible,three nucleotide sequences may be joined by the use of a linker moleculewhich contains topoisomerases at both the 5′ and 3′ termini of each end.Thus, upon joining of the three nucleotide sequences, a singlenucleotide sequence is formed which contains no nicks at the junctionpoints. This process has the advantage of employing a singletopoisomerase modified molecule to join three nucleotide sequencestogether.

The present invention also provides compositions, and kits containingsuch compositions, including kits containing component useful forperforming methods of the invention. In one aspect, a composition of theinvention comprises isolated components characteristic of a step of amethod of the invention. For example, a composition of the invention cancomprise two or more of the same or different topoisomerase-charged dsnucleotide sequences. As used herein, the term “different,” when used inreference to the ds nucleotide sequences of a composition of theinvention, means that the ds nucleotide sequences share less than 95%sequence identity with each when optimally aligned, generally less than90% sequence identity, and usually less than 70% sequence identity.Thus, ds nucleotide sequences that, for example, differ only in beingpolymorphic variants of each other or that merely contain different 5′or 3′ overhanging sequences are not considered to be “different” forpurposes of a composition of the invention. In comparison, different dsnucleotide sequences are exemplified by a first sequence encoding apolypeptide and second sequence comprising a regulatory element, or afirst sequence encoding a first polypeptide a second sequence encoding anon-homologous polypeptide.

Where a composition of the invention comprises more than two differentisolated ds nucleotide sequences or more than two differenttopoisomerase-charged ds nucleotide sequences, each of the ds nucleotidesequences is different from each other, i.e., they are all differentfrom each other. However, it will be recognized that each of the dsnucleotide sequences, for example, a sequence referred to as a first dsnucleotide sequence, generally comprises a population of such nucleotidesequences, which are identical or substantially identical to each other.Thus, it should be clear that the term “different” is used in comparing,for example, a first (or population of first) ds nucleotide sequenceswith a second (and other) ds nucleotide sequence. A compositioncomprising two or more different topoisomerase-charged ds nucleotidesequences can further comprise a topoisomerase. Examples of such dsnucleotide sequences comprising the components of a composition of theinvention are disclosed herein and include, for example, codingsequences, transcriptional regulatory element, translational regulatoryelements, elements encoding a detectable or selectable markers such asan epitope tag or an antibiotic resistance gene, elements encodingpolypeptide domains such as cell compartmentalization domains or signalpeptides, and the like.

As used herein, the term “isolated” means that a molecule being referredto is in a form other than that in which it exists in nature. Ingeneral, an isolated nucleotide sequence, for example, can be anynucleotide sequence that is not part of a genome in a cell, or isseparated physically from a cell that normally contains the nucleotidesequence. It should be recognized that various compositions of theinvention comprise a mixture of isolated ds nucleotide sequences. Assuch, it will be understood that the term “isolated” only is used inrespect to the isolation of the molecule from its natural state, butdoes not indicate that the molecule is an only constituent.

A composition of the invention can comprise two different ds nucleotidesequences, each of which contains a topoisomerase recognition site at ornear one or both ends, and a site specific topoisomerase, which can bindto and cleave the ds nucleotide sequences at the topoisomeraserecognition site. Optionally, at least one of the different dsnucleotide sequences can be a topoisomerase-charged ds nucleotidesequence. Preferably, the topoisomerase covalently bound to thetopoisomerase-charge ds nucleotide sequence is of the same family as thetopoisomerase in the composition.

Various combinations of components can be used in a method of theinvention. For example, the method can be performed by contacting atopoisomerase-activated first ds nucleotide sequence; a second dsnucleotide sequence having a first end and a second end, wherein at thefirst end or second end or both, the second nucleotide sequence has atopoisomerase recognition site at or near the 3′ terminus, and ahydroxyl group at the 5′ terminus of the same end; and a topoisomerase.Where the 5′ terminus of one or both ends to be linked has a 5′phosphate group, a phosphatase also can be contacted with the componentsof the reaction mixture. Upon such contacting, the topoisomerase cancleave the second nucleotide sequence to produce atopoisomerase-activated second ds nucleotide sequence, the phosphatase,if necessary, can generate a 5′ hydroxyl group at the same end, and thesecond ds nucleotide sequence then can be covalently linked to thetopoisomerase-activated first ds nucleotide sequence. As such, it willbe recognized that a composition of the invention can comprise any ofvarious combinations of components useful for performing a method of theinvention.

In general, a method of the invention for generating a ds recombinantnucleic acid molecule covalently linked in both strands is based on thedetermination that a ds recombinant nucleic acid molecule covalentlylinked in both strands can be produced by contacting a first dsnucleotide sequence with a second ds nucleotide sequence, wherein thefirst and second sequences each have, at the ends to be linked, atopoisomerase recognition site, for example, 5′-(C/T)CCTT-3′ (Shuman,supra, 1991; U.S. Pat. No. 5,766,891). Upon cleavage, the site specifictopoisomerase is covalently bound at the 3′ terminus. Where the cleavednucleotide sequences also contain a 5′ hydroxy group at the same end asthe bound topoisomerase, and the ends of the two nucleotide sequencesassociate, the topoisomerase on each 3′ terminus can covalently linkthat terminus to a 5′ hydroxyl group on the associated nucleotidesequence (see FIG. 1).

As used herein, reference to contacting a first nucleotide sequence andat least a second nucleotide sequence “under conditions such that allcomponents are in contact” means that the reaction conditions areappropriate for the topoisomerase-cleaved ends of the nucleotidesequences to come into sufficient proximity such that a topoisomerasecan effect its enzymatic activity and covalently link the 3′ or 5′terminus of a first nucleotide sequence to a 5′ or 3′ terminus,respectively, of a second nucleotide sequence. Examples of suchconditions, which include the reaction temperature, ionic strength, pH,and the like, are disclosed herein, and other appropriate conditions asrequired, for example, for particular 5′ overhanging sequences of thetermini generated upon topoisomerase cleavage, can be determinedempirically or using formulas that predict conditions for specifichybridization of nucleotide sequences, as is well known in the art (see,for example, (Sambrook et al., Molecular Cloning: A laboratory manual(Cold Spring Harbor Laboratory Press 1989); Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1987, and supplements through 1995), each of which is incorporatedherein by reference).

In one embodiment, a method of the invention provides a means to renderan open reading from a cDNA or an isolated genomic DNA sequenceexpressible by operatively linking one or more regulatory elements tothe putative coding sequence. Accordingly, a first ds nucleotidesequence comprising an open reading frame can be amplified by PCR usinga primer pair that generates an amplified first ds nucleotide sequencehaving a topoisomerase recognition site at one or both ends, as desired,preferably such that, upon cleavage by the site specific topoisomerase,one or both ends contains a defined 5′ or 3′ overhang. Where both endsof the amplified first ds nucleotide sequence are so constructed, the 5′or 3′ overhanging sequences generally, but not necessarily, aredifferent from each other. The amplified first ds nucleotide sequencethen can be contacted with a second ds nucleotide sequence comprising adesired regulatory element such as a promoter and, in certainembodiments, a topoisomerase recognition site, and with a topoisomerase,such that the second nucleotide sequence is operatively covalentlylinked to the 5′ end of the coding sequence according to a method of theinvention.

In such a method, a second (or other) ds nucleotide sequence also cancomprise two or more regulatory elements, for example, a promoter, aninternal ribosome entry site and an ATG initiator methionine codon, orthe like, or other sequence of interest, for example, an sequenceencoding an epitope tag, in operative linkage with each other, and whichcan be operatively covalently linked to the 5′ end of a first dsnucleotide sequence comprising a coding sequence. Such a method canfurther include contacting a third ds nucleotide sequence comprising,for example, a polyadenylation signal, which can be operativelycovalently linked according to a method of the invention to the 3′ endof the coding sequence, thereby generating an expressible ds recombinantnucleic acid molecule. As such, a method of the invention provides ameans for generating a functional ds recombinant nucleic acid moleculethat can be transcribed, translated, or both as a functional unit. Asdisclosed herein, the inclusion of complementary 5′ or 3′ overhangingsequences generated by topoisomerase cleavage at the termini of the dsnucleotide sequences to be linked together by the site specifictopoisomerase facilitates the generation of a ds recombinant nucleicacid molecule having a desired directional orientation of the nucleotidesequences in the construct.

In another embodiment, a method of the invention is performed such thatthe first ds nucleotide sequence or a second (or other) ds nucleotidesequence, or combination thereof, is one of a plurality of nucleotidesequences. As used herein, the term “plurality,” when used in referenceto a first or at least a second nucleotide sequence, means that thenucleotide sequences are related but different. For purposes of thepresent invention, the nucleotide sequences of a plurality are “related”in that each nucleotide sequence in the plurality contains at least atopoisomerase recognition site, or a cleaved form thereof, at one ormore termini. Furthermore, the nucleotide sequences of a plurality are“different” in that they can comprise, for example, a cDNA library, acombinatorial library of nucleotide sequences, a variegated populationof nucleotide sequences, or the like. Methods of making cDNA libraries,combinatorial libraries, libraries comprising variegated populations ofnucleotide sequences, and the like are well known in the art (see, forexample, U.S. Pat. No. 5,837,500; U.S. Pat. No. 5,622,699; U.S. Pat. No.5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al.,Gene 109:13-19,1991; O'Connell et al., Proc. Natl. Acad. Sci., USA93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold etal., Ann. Rev. Biochem. 64:763-797, 1995; each of which is incorporatedherein by reference).

The present invention further provides a method of generating a dsrecombinant nucleic acid molecule covalently linked in both strands byamplifying a portion of a first nucleotide sequence using a PCR primerpair, wherein at least one primer of the primer pair encodes atopoisomerase recognition site or a complement thereof, therebyproducing a first ds nucleotide sequence having a first end and a secondend, wherein the first end or second end or both has a topoisomeraserecognition site at the 3′ terminus and/or the 5′ terminus; andcontacting the first ds nucleotide sequence; at least a second dsnucleotide sequence having a first end and a second end, wherein thefirst end or second end or both has a topoisomerase recognition site atthe 3′ terminus and/or the 5′ terminus, or a cleavage product thereof;and a topoisomerase (see FIG. 1). When contacted under conditions suchthat an end of the first ds nucleotide sequence having a topoisomeraserecognition site and an end of the at least second ds nucleotidesequence having a topoisomerase recognition site can associate, a dsrecombinant nucleic acid molecule covalently linked in both strands isgenerated.

As disclosed herein, a PCR method using primers designed to incorporatea topoisomerase recognition site at one or both ends of an amplified dsnucleotide sequence provides a convenient means for producing dsnucleotide sequences useful in a method of the invention. In certainembodiments, at least one of the primers of a primer pair is designedsuch that it comprises, in a 5′ to 3′ orientation, a nucleotide sequencecomplementary to a topoisomerase recognition site, and a nucleotidesequence complementary to the 3′ end of a target nucleic acid moleculeto be amplified (i.e., a target specific region). In addition, theprimer can contain, in a position 5′ to the complement of thetopoisomerase recognition site, a desired nucleotide sequence of anylength (generally about 1 to 100 nucleotide, usually about 2 to 20nucleotides, and particularly about 4 to 12 nucleotides), which, uponcleavage of the amplification product by a site specific topoisomerase,forms a desired 5′ overhang. The second primer of the PCR primer paircan be complementary to a desired sequence of the nucleotide sequence tobe amplified, and can comprise a complement to a topoisomeraserecognition site, a sequence that would generate a 5′ overhang uponcleavage by a site specific topoisomerase, or any other sequence, asdesired.

Such a primer can comprise or encode any other sequence of interest,including, for example, a site specific integration recognition sitesuch as an att site, a lox site, or the like, or, as discussed above,can simply be used to introduce a topoisomerase recognition site into ads nucleotide sequence comprising such a sequence of interest. A dsrecombinant nucleic acid molecule generated according to a method of theinvention and containing a site specific integration recognition sitesuch as an att site or lox site can be integrated specifically into adesired locus such as into a vector, a gene locus, or the like, thatcontains the required integration site, for example, an att site or loxsite, respectively, and upon contact with the appropriate enzymesrequired for the site specific event, for example, lambda Int and IHFproteins or Cre recombinase, respectively. The incorporation, forexample, of attB or attP sequences into a ds recombinant nucleic acidmolecule covalently linked in both strands according to a method of theinvention allows for the convenient manipulation of the nucleic acidmolecule using the GATEWAY™ Cloning System (Invitrogen Corp., La JollaCalif.).

In one embodiment, a construct generated according to a method of theinvention is further amplified by a PCR reaction or other amplificationreaction. Direct PCR of a ds recombinant nucleic acid molecule generatedaccording to a method of the invention is possible because the constructis covalently linked in at least one strand. As such, PCR can be used togenerate a large amount of the construct. More importantly, as indicatedabove, PCR provides an in vitro selection method for obtaining only adesired product generated according to a method of the invention,without obtaining partial reaction products. For example, a method ofthe invention can be used to generate a ds recombinant nucleic acidmolecule covalently linked in both strands comprising, operativelylinked in a 5′ to 3′ orientation, a first ds nucleotide sequencecomprising a promoter, a second ds nucleotide sequence comprising acoding region, and a third ds nucleotide sequence comprising apolyadenylation signal.

As disclosed herein, a construct having a predetermined orientation canbe generated by including complementary 5′ overhanging sequences on theends of the ds nucleotide sequences to be joined. By selecting a PCRprimer pair including a first primer complementary to the first dsnucleotide sequence and upstream of the promoter sequence, and a secondprimer complementary to the third ds nucleotide sequence and downstreamof the polyadenylation signal, a functional amplification productcomprising the promoter, coding region and polyadenylation signal can begenerated. In contrast, partial reaction products that lack either thefirst ds nucleotide sequence or third ds nucleotide is not amplifiedbecause either the first or second primer, respectively, would nothybridize to the partial product. In addition, a construct lacking thesecond ds nucleotide sequence would not be generated due to the lack ofcomplementarity of the 5′ overhanging sequences of the first and thirdds nucleotide sequences. As such, a method of the invention provides ameans to obtain a desired functional ds recombinant nucleic acidmolecule covalently linked in both strands.

The use of PCR in such a manner further provides a means to screen alarge number of nucleic acid molecules generated according to a methodof the invention in order to identify constructs of interest. Sincemethods for utilizing PCR in automated high throughput analyses areroutine and well known, it will be recognized that the methods of theinvention can be readily adapted to use in a high throughput system.Using such a system, a large number of constructs can be screened inparallel, and partial or incomplete reaction products can be identifiedand disposed of, thereby preventing a waste of time and expense thatwould otherwise be required to characterize the constructs or examinethe functionality of the constructs in further experiments.

The methods of the invention have broad application to the field ofmolecular biology. As discussed in greater detail below, the methods ofthe invention can be used, for example, to label DNA or RNA probes, toperform directional cloning (see Example 1.B), to generate sense orantisense RNA molecules (see Example 2.A), to prepare bait or preyconstructs for performing a two hybrid assay (see Example 2.C), toprepare linear expression elements (see Examples 2.A and 2.B), and toprepare constructs useful for coupled in vitro transcription/translationassays (see Example 2.B). For example, a method of generating dsrecombinant nucleic acid molecules covalently linked in both strandsprovides a means to generate linear expression elements (LEEs), whichconsist of a linear nucleic acid molecule comprising two or morenucleotide sequences such as a promoter or other regulatory elementlinked to an open reading frame (see Example 1). LEEs have been reportedto efficiently transfect cells, thus bypassing a requirement for cloningthe expression element in a vector (Sykes and Johnston, Nat. Biotechnol.17:355-359, 1999). The components of a LEE can be noncovalently linked,or can be covalently linked via a ligation reaction. The preparation ofnoncovalently linked LEEs requires using PCR primers containingdeoxyuridine residues to amplify each nucleotide sequence component,then treating the PCR products with uracil-DNA glycosylase to generateoverhanging ends that can hybridize. However, the efficiency oftransfection using such noncovalently linked LEEs is variable, and, insome cases, much lower than the efficiency of covalently linked LEEs(Sykes and Johnston, supra, 1999). Furthermore, such LEEs are notsuitable for use as templates for PCR amplification because the primerextension reaction cannot proceed past nicks in the template and,therefore, is terminated producing incomplete reaction products.

A method of the invention provides a straightforward and simple means togenerate covalently linked LEEs, thereby avoiding the inconvenient andadditional steps previously described for preparing a LEE, as well asreducing variability in transfection efficiency as observed usingnoncovalently linked LEEs. For example, a first ds nucleotide sequence,which encodes an open reading frame of interest, can be amplified by PCRas disclosed herein to contain a topoisomerase recognition site, orcleavage product thereof, on one or both ends. Furthermore, the PCRprimers can be designed such that, upon cleavage of the amplified firstds nucleotide sequence by a site specific topoisomerase, the cleavageproduct contains a predetermined and desired 5′ overhanging sequence. Asecond nucleotide sequence (and a third or more, as desired), inaddition to containing a topoisomerase recognition site, or cleavageproduct thereof, can include or encode a regulatory element, forexample, a promoter, an enhancer, a silencer, a splice acceptor site, atranslation start site, a ribosome recognition site or internal ribosomeentry site, a polyadenylation signal, an initiator methionine codon, ora STOP codon, or can encode any other desired sequence such as anepitope tag or cell compartmentalization domain. Preferably, the second(or other) ds nucleotide sequence to be covalently linked to the firstds nucleotide sequence has a 5′ overhanging sequence that iscomplementary to the 5′ overhang at the end of the first ds nucleotidesequence to which it is to be linked. Upon contact of such nucleotidesequences in presence of a topoisomerase a promoter, for example, can beoperatively covalently linked to the 5′ terminus of the open readingframe, and a polyadenylation signal can be operatively covalently linkedto the 3′ terminus of the open reading frame, thereby generating acovalently linked functional LEE (see Example 1).

Examples of regulatory elements useful in the present invention aredisclosed herein and include transcriptional regulatory elements,translational regulatory elements, elements that facilitate thetransport or localization of a nucleotide sequence or polypeptide in (orout of) a cell, elements that confer a detectable phenotype, and thelike. Transcriptional regulatory elements include, for example,promoters such as those from cytomegalovirus, Moloney leukemia virus,and herpes virus, as well as those from the genes encodingmetallothionein, skeletal actin, phosphoenolpyruvate carboxylase,phosphoglycerate, dihydrofolate reductase, and thymidine kinase, as wellas a GAL4 promoter and promoters from viral long terminal repeats (LTRs)such as Rous sarcoma virus LTR; enhancers, which can be constitutivelyactive such as an immunoglobulin enhancer, or inducible such as SV40enhancer; and the like. For example, a metallothionein promoter is aconstitutively active promoter that also can be induced to a higherlevel of expression upon exposure to a metal ion such as copper, nickelor cadmium ion. In comparison, a tetracycline (tet) inducible promoteris an example of a promoter that is induced upon exposure totetracycline, or a tetracycline analog, but otherwise is inactive. Atranscriptional regulatory element also can be a tissue specificregulatory element, for example, a muscle cell specific regulatoryelement, such that expression of an encoded product is restricted to themuscle cells in an individual, or to muscle cells in a mixed populationof cells in culture, for example, an organ culture. Muscle cell specificregulatory elements including, for example, the muscle creatine kinasepromoter (Sternberg et al., Mol. Cell. Biol. 8:2896-2909, 1988, which isincorporated herein by reference) and the myosin light chainenhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA88:5847-5851, 1991, which is incorporated herein by reference) are wellknown in the art. Other tissue specific promoters, as well as regulatoryelements only expressed during particular developmental stages of a cellor organism are well known in the art.

Regulatory or other elements useful in generating a construct accordingto a method of the invention can be obtained in various ways. Inparticular, many of the elements are included in commercially availablevectors and can be isolated therefrom and can be modified to contain atopoisomerase recognition site at one or both ends, for example, using aPCR method as disclosed herein. In addition, the sequences of orencoding the elements useful herein generally are well known anddisclosed in publications. In many cases, the elements, for example,many transcriptional and translational regulatory elements, as well ascell compartmentalization domains, are relatively short sequences and,therefore, are amenable to chemical synthesis of the element or anucleotide sequence encoding the element. Thus, in one embodiment, anelement comprising a composition of the invention, useful in generatinga ds recombinant nucleic acid molecule according to a method of theinvention, or included within a kit of the invention, can be chemicallysynthesized and, if desired, can be synthesized to contain atopoisomerase recognition site at one or both ends of the element and,further, to contain an overhanging sequence following cleavage by a sitespecific topoisomerase.

A topoisomerase-charged vector can be generated in the following manner(Genome Res. 9: 383-392, 1999): A vector is linearized with arestriction enzyme that leaves “sticky ends”. Using a ligase such as T4DNA ligase, adapter oligonucleotides are ligated to both ends, and bothstrands, of the linearized DNA. The adapter oligonucleotides contain andposition a 5′-CCCTT-3′ Vaccinia topoisomerase type I recognitionsequence such that it can be cleaved by topoisomerase and trap thecovalent topoisomerase-DNA complex at each 3′ end of the vector. Theadapted vector is then incubated with purified Vaccinia topoisomeraseand an annealing oligonucleotide that complete the “topoisomerase sites”at each end of the vector. The annealing oligonucleotide acts to leave abreak, or nick, in the “bottom” strand opposite the last T in the5′-CCCTT-3′ containing oligonucleotide. The oligonucleotide adapterfragments that are “downstream” of the topoisomerase cleavage site (the“leaving groups”) are released upon topoisomerase cleavage and areremoved in the topoisomerase-vector purification process. In the absenceof the 5′ hydroxyl from the “leaving group”, topoisomerase is trapped ina covalent complex with the DNA ends to produce a topoisomerase-chargedvector.

Where ds nucleotide sequences are to be covalently linked according to amethod of the invention, the nucleotide sequences generally areoperatively linked such that the recombinant nucleic acid molecule thatis generated has a desired structure and performs a desired function orencodes a desired expression product. As used herein, the terms“operatively linked,” and “operably connected,” or the like, mean thattwo or more nucleotide sequences are positioned with respect to eachother such that they act as a unit to effect a function attributable toone or both sequences or a combination thereof. The term “operativelycovalently linked” is used herein to refer to operatively linkednucleotide sequences generated according to a method of the inventionfor generating a ds recombinant nucleic acid molecule covalently linkedin one or both strands. For example, a nucleotide sequence containing anopen reading frame can be operatively linked to a promoter such that thepromoter confers its regulatory effect on the open reading framesimilarly to the way in which it would effect expression of an openreading frame that it normally is associated with in a genome in a cell.Similarly, two or more nucleotide sequences comprising open readingframes can be operatively linked in frame such that, upon transcriptionand translation, a chimeric fusion polypeptide is produced.

Although a ds recombinant nucleic acid molecule covalently linked in oneor both strands, generated according to a method of the inventiongenerally is linear, the construct generated also can be a circularizedds recombinant nucleic acid molecule. Furthermore, a circular dsrecombinant nucleic acid molecule can be generated such that it has thecharacteristics of a vector, and contains, for example, regulatoryelements (expression control sequences) required for replication in aprokaryotic host cell, a eukaryotic host cell, or both; can contain anucleotide sequence encoding a polypeptide that confers antibioticresistance; a multiple cloning site; or the like. An advantage of such amethod is that the generated ds recombinant nucleic acid molecule, whichis circularized according to a method of the invention, can betransformed or transfected into an appropriate host cell, wherein theconstruct is amplified. Thus, in addition to an in vitro method such asPCR, which can be used to generate large amounts of a linear dsrecombinant nucleic acid molecule generated according to a method of theinvention, an in vivo method using a host cell can be used for obtaininga large amount of a circularized product generated according to a methodof the invention. Such elements including bacterial origins ofreplication, antibiotic resistance genes, and the like, which comprise atopoisomerase recognition site according to the present invention, canbe useful components to include in a kit of the invention as disclosedherein.

It should be recognized that a linear ds recombinant nucleic acidmolecule covalently linked in one or both strands, also can be clonedinto a vector, which can be a plasmid vector or a viral vector such as abacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vacciniavirus, semliki forest virus and adeno-associated virus vector, all ofwhich are well known and can be purchased from commercial sources(Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL,Gaithersburg Md.). If desired, the vector can be linearized and modifiedaccording to a method of the invention, for example, using a PCR method,to contain a topoisomerase recognition site, or cleavage productthereof, at one or both 3′ termini, or can be constructed by one skilledin the art (see, generally, Meth. Enzymol., Vol. 185, Goeddel, ed.(Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994;Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J.Clin. Invest. 92:381-387, 1993; each of which is incorporated herein byreference).

Viral expression vectors can be particularly useful where a method ofthe invention is practiced for the purpose of generating a dsrecombinant nucleic acid molecule covalently linked in one or bothstrands, that is to be introduced into a cell, particularly a cell in asubject. Viral vectors provide the advantage that they can infect hostcells with relatively high efficiency and can infect specific cell typesor can be modified to infect particular cells in a host.

Viral vectors have been developed for use in particular host systems andinclude, for example, baculovirus vectors, which infect insect cells;retroviral vectors, other lentivirus vectors such as those based on thehuman immunodeficiency virus (HIV), adenovirus vectors, adeno-associatedvirus (AAV) vectors, herpesvirus vectors, vaccinia virus vectors, andthe like, which infect mammalian cells (see Miller and Rosman,BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl.,1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J.Med. 334:1185-1187 (1996), each of which is incorporated herein byreference). For example, a viral vector based on an HIV can be used toinfect T cells, a viral vector based on an adenovirus can be used, forexample, to infect respiratory epithelial cells, and a viral vectorbased on a herpesvirus can be used to infect neuronal cells. Othervectors, such as AAV vectors can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

The present invention also provides methods for preparing recombinantnucleic acid molecules containing viral nucleic acid sequences, as wellas covalently linked recombinant nucleic acid molecules prepared by suchmethods and compositions containing the recombinant nucleic acidmolecules. Viral vectors derived from adenoviruses, for example, havebeen used for introducing expressible polynucleotides into cells,including in methods of gene therapy. Adenoviral vectors areparticularly attractive vehicles for delivering genes into respiratoryepithelial cells. Adenoviruses naturally infect respiratory epitheliawhere they cause a mild disease. Other targets for adenovirus-baseddelivery systems are liver, the central nervous system, endothelialcells, and muscle. Adenoviruses have the advantage of being capable ofinfecting non-dividing cells (see Kozarsky and Wilson, Curr. Opin.Genet. Develop. 3:499-503, 1993, presenting a review of adenovirus-basedgene therapy; Bout et al., Human Gene Ther. 5:3-10, 1994, demonstratingthe use of adenovirus vectors to transfer genes to the respiratoryepithelia of rhesus monkeys; see, also, Rosenfeld et al., Science252:431-434, 1991; Rosenfeld et al., Cell 68:143-155, 1992; Mastrangeliet al., J. Clin. Invest. 91:225-234, 1993; Internatl. Publ. Nos.WO94/12649 and WO 96/17053; U.S. Pat. No. 5,998,205; and Wang et al.,Gene Ther. 2:775-783, 1995, each of which is incorporated herein byreference. Accordingly, the present invention provides methods ofgenerating vectors containing adenoviral sequences, and further providesmethods of using such adenoviral vectors for introducing apolynucleotide into cells such as respiratory epithelial cells.

Viral vectors derived from adeno-associated viruses (AAV) andherpesviruses also can be used for introducing a polynucleotide intocells, particularly mammalian cells, in vitro and in vivo, for example,for a gene therapy procedure (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300,1993; U.S. Pat. No. 5,436,146; Wagstaffet al., Gene Ther.5:1566-70, 1998, each of which is incorporated herein by reference). Forexample, viral vectors derived from herpesvirus are particularly usefulfor applications where it is desired to introduce and express apolynucleotide in nerve cells. Accordingly, the present invention alsoprovides methods of generating vectors containing herpesvirus or AAVnucleotide sequences, and further provides methods of using such viralvectors for introducing a polynucleotide into cells.

As such, the present invention provides methods for preparingrecombinant nucleic acid molecules having one or more functionalproperties of viral vectors (e.g., adenoviral vectors, alphaviralvectors, herpes viral vectors, AAV vectors, etc.). In particularembodiments, methods of the invention include the covalently linkingnucleotide sequences, wherein one or more of the nucleotide sequencescontains regions of a viral genome that confer a function characteristicof the virus from which the nucleotide sequence was derived, forexample, the ability to replicate in one or few specific host cells, theability to be packaged into viral particles, and the like.

In particular embodiments, the invention includes methods for preparingadenoviral vectors by covalently linking at least one (e.g., one, two,three, four, etc.) nucleotide sequence comprising adenoviral sequencesto one or more other nucleotide sequences. Specific examples ofadenoviral vectors and nucleotide sequences that can be used to prepareadenoviral vectors are disclosed in U.S. Pat. Nos. 5,932,210, 6,136,594,and 6,303,362, each of which is incorporated herein by reference.Adenoviral vectors prepared by methods of the invention can bereplication competent or replication deficient. For example, when areplication deficient adenoviral vector is desired, the adenoviralnucleotide sequence can contain deletions of all or part of one or moreof the E1a region, the E1b region, and the E3 region. Adenoviral vectorscontaining deletions of these regions are described, for example, inU.S. Pat. No. 6,136,594. Accordingly, adenoviral vectors prepared bymethods of the invention are provided, as are compositions containingthe vectors, and uses of such vectors, for example, use of theadenoviral vectors to deliver a heterologous polynucleotide to cells ofa mammal (e.g., a human). Thus, the invention provides methods forpreparing vectors suitable for use in gene therapy protocols. Typically,such vectors are replication deficient.

In specific embodiments, adenoviral vectors of the invention comprisesubstantially the entire adenoviral genome, except that one or more ofthe E1a region, the E1b region, and the E3 region are deleted. Infurther specific embodiments, non-adenoviral nucleotide sequences can bepresent in one or more of the E1a region, the E1b region, and the E3region. In particular embodiments, adenoviral vectors prepared bymethods of the invention contain at least one origin of replicationand/or a selection marker, for example, a prokaryotic origin ofreplication, which allows for amplification of the vector in prokaryoticcells such as E. coli cells.

As described above, AAV and herpesvirus vectors also can be preparedaccording to the methods of the invention. In addition, the alphaviralvectors (e.g., Sindbis virus vectors, Semliki Forest virus vectors, RossRiver virus vectors, Venezuelan equine encephalitis virus vectors,Western equine encephalitis virus vectors, Eastern equine encephalitisvirus vectors, etc.) can be prepared according to a method of theinvention. As such, the present invention provides herpesvirus vectors,AAV vectors, alphaviral vectors, and the like, prepared by such methods,compositions containing such viral vectors, and methods of using theviral vectors.

In particular embodiments, the invention includes methods for preparingalphaviral vectors by covalently linking at least one nucleotidesequence comprising alphaviral sequences to one or more other nucleotidesequences. Specific examples of alphaviral vectors and nucleotidesequences thereof useful for preparing alphaviral vectors are describedin U.S. Pat. Nos. 5,739,026 and 6,224,879; Gibco/BRL Instruction ManualNo. 10179-018, “SFV Gene Expression System” (Gaithersburg Md.); andInvitrogen Sindbis Expression System manual, catalog no. K750-01(version E; Carlsbad Calif.), each of which is incorporated herein byreference. In specific embodiments, alphavirus nucleotide sequences usedin methods of the invention to prepare alphaviral vectors contain one ormore packaging signals, which can, but need not, be of alphaviralorigin; one or more subgenomic promoters; one or more nucleotidesequences encoding a non-structural protein such as nsp1, nsp2, nsp3,nsp4, etc.; and combinations thereof.

Alphaviral vectors of the invention can be introduced into cells as DNAor RNA molecules. When DNA forms of the vectors are introduced intocells, expression control sequences (e.g., inducible, repressible orconstitutive expression control sequences) can be used to generate RNAmolecules, from which one or more non-structural proteins can betranslated. In specific embodiments, the non-structural proteins form anRNA dependent RNA polymerase that can amplify RNA moleculescorresponding to all or a portion of the transcript generated from theDNA form of the alphaviral vector. As such, these non-structuralproteins can catalyze the production of additional copies of RNAmolecules from RNA templates, resulting in RNA amplification. Further,one or more nucleotide sequences, for which high levels of expressionare desired, can be operatively linked to a subgenomic promoter, thusresulting in the production of high levels of RNA corresponding to theone or more nucleotide sequences.

In an exemplary embodiment, alphaviral vectors prepared by methods ofthe invention comprise DNA, wherein an inducible promoter directstranscription of an RNA molecule encoding nsp1, nsp2, nsp3, and nsp4 ofa Sindbis virus, and wherein a Sindbis subgenomic promoter isoperatively linked to a nucleotide sequence that is not of Sindbis viralorigin. The invention also provides alphaviral vectors prepared bymethods of the invention, methods of using such alphaviral vectors, andcompositions containing such alphaviral vectors.

The invention further provides methods for covalently linking nucleotidesequences, wherein one or more of the nucleotide sequences contains oneor more (e.g., one, two, three, four, etc.) viral packaging signals(e.g., one or more packaging signal derived from a virus referred toabove). The presence of such packaging signals directs the packaging ofthe recombinant nucleic acid molecule viral vector prepared by methodsof the invention. One method for preparing packaged viral vectors is byintroducing or expressing the viral vectors, which are preparedaccording to a method of the invention, into packaging cell lines, whichexpress proteins suitable for the production of virus-like particles.Accordingly, the invention provides packaged recombinant nucleic acidmolecules of the invention, methods for preparing such packaged nucleicacid molecules, and compositions containing the packaged nucleic acidmolecules.

It will be recognized that a nucleotide sequence to be covalently linkedto one or more other nucleotide sequences according to a method of theinvention can be any nucleotide sequence, and generally is a nucleotidesequence providing some desirable structural or functional feature tothe covalently linked recombinant nucleic acid molecule generatedthereby. For example, the nucleotide sequence can contain a restrictionendonuclease site or recombinase recognition site, or can comprise amultiple cloning site, which contains two or more restrictionendonuclease site or recombinase recognition site or combinationsthereof. As such, the present invention also provides methods forpreparing a covalently linked recombinant nucleic acid moleculecontaining one or more (e.g., one, two, three, four, five, six, etc.)multiple cloning sites, which can be the same or different, and can beadjacent to each other or separated by one or more other nucleotidesequences in the covalently linked recombinant nucleic acid molecule.Thus, one or more nucleotide sequences used in a method of the inventioncan comprise one or more multiple cloning sites. One or more multiplecloning sites also can be added to nucleotide sequences used to preparethe recombinant nucleic acid molecules, for example, by attachinglinkers that contain the one or more multiple cloning sites. In relatedaspects, the invention includes recombinant nucleic acid molecules thatare prepared by methods of the invention and contain one or moremultiple cloning sites, as well as the use of one or more these multiplecloning sites to modify recombinant nucleic acid molecules prepared bymethods of the invention. The invention also provides recombinantnucleic acid molecules produced by such a method, as well as uses ofthese molecules and compositions containing these molecules. In oneembodiment, the generated recombinant nucleic acid molecule furthercomprises nucleotides sequences that allow the recombinant nucleic acidmolecule to function as a vector, for example, viral nucleotidesequences such as adenovirus, herpesvirus, retrovirus, AAV, oralphavirus nucleotide sequences.

Nucleotide sequences useful in a method of the invention also can alsocomprise or encode one or more operators. Operators are well known inthe art and include, for example, the tryptophan operator of thetryptophan operon of E. coli. The tryptophan repressor, when bound totwo molecules of tryptophan, binds to the E. coli tryptophan operatorand, when suitably positioned (i.e., operatively linked) with respect tothe promoter, blocks transcription. Another example of an operatorsuitable for use with the invention is operator of the E. colitetracycline operon. Components of the tetracycline resistance system ofE. coli can function in eukaryotic cells and are useful for regulatinggene expression in eukaryotic cells, for example, mammalian cells suchas human cells. The tetracycline repressor, which binds to tetracyclineoperator in the absence of tetracycline and represses genetranscription, also has been expressed in plant cells at sufficientlyhigh concentrations to repress transcription from a promoter containingtetracycline operator sequences (Gatz et al., Plants 2:397-404, 1992).Tetracycline regulated expression systems are described, for example inU.S. Pat. No. 5,789,156, which is incorporated herein by reference.Additional examples of operators that can be used in a method or togenerate a composition of the invention include the Lac operator and theoperator of the molybdate transport operator/promoter system of E. coli(see, for example, Cronin et al., Genes Devel. 15:1461-1467, 2001;Grunden et al., J. Biol. Chem. 274:24308-24315, 1999, each of which isincorporated herein by reference).

Thus, in particular embodiments, the invention provides methods forpreparing covalently linked recombinant nucleic acid molecules thatcontain one or more operators, which can be used to regulate expressionof an operatively linked expressible polynucleotide in prokaryotic cellsor eukaryotic cells. As will be recognized, when such a recombinantnucleic acid molecule, which contains an operator, is placed underconditions in which transcriptional machinery is present, either in vivoor in vitro, regulation of expression of an operatively linkedpolynucleotide can be modulated by contacting the nucleic acid moleculewith a repressor and one or more metabolites that facilitate binding ofan appropriate repressor to the operator. Accordingly, the presentinvention further provides methods for preparing covalently linkedrecombinant nucleic acid molecules that encode one or more repressors,which modulate the function of operators, as well as the recombinantnucleic acid molecules produced by such methods, compositions containingthe recombinant nucleic acid molecules, and uses of the recombinantnucleic acid molecules and the compositions.

A method of the invention can be used to operatively covalently link afirst ds nucleotide sequence containing an open reading frame to asecond (and other) ds nucleotide sequence containing an open readingframe such that a nucleic acid molecule encoding a chimeric polypeptideis generated. The chimeric polypeptide comprises a fusion polypeptide,in which the two (or more) encoded peptides (or polypeptides) aretranslated into a single product, i.e., the peptides are covalentlylinked through a peptide bond. For example, a first ds nucleotidesequence can encode a cell compartmentalization domain, such as a plasmamembrane localization domain, a nuclear localization signal, amitochondrial membrane localization signal, an endoplasmic reticulumlocalization signal, or the like, or a protein transduction domain suchas the human immunodeficiency virus TAT protein transduction domain,which can facilitate translocation of a peptide linked thereto into acell (see Schwarze et al., Science 285:1569-1572, 1999; Derossi et al.,J. Biol. Chem. 271:18188, 1996; Hancock et al., EMBO J. 10:4033-4039,1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No.5,776,689 each of which is incorporated herein by reference). Such adomain can be useful to target a fusion polypeptide comprising thedomain and a polypeptide encoded by a second ds nucleotide sequence, towhich it is covalently linked according to a method of the invention, toa particular compartment in the cell, or for secretion from or entryinto a cell. As such, the invention provides a means to generate dsrecombinant nucleic acid molecules covalently linked in both strandsthat encode a chimeric polypeptide.

A fusion polypeptide expressed from a nucleic acid molecule generatedaccording to a method of the invention also can comprise a peptidehaving the characteristic of a detectable label or a tag such that theexpress fusion polypeptide can be detected, isolated, or the like. Forexample, a ds nucleotide sequence containing a topoisomerase recognitionsite, or cleavage product thereof, as disclosed herein, can encode anenzyme such as alkaline phosphatase, β-galactosidase, chloramphenicolacetyltransferase, luciferase, or other enzyme; or can encode a peptidetag such as a polyhistidine sequence (e.g., hexahistidine), a V5epitope, a c-myc epitope; a hemagglutinin A epitope, a FLAG epitope, orthe like. Expression of a fusion polypeptide comprising a detectablelabel can be detected using the appropriate reagent, for example, bydetecting light emission upon addition of luciferin to a fusionpolypeptide comprising luciferase, or by detecting binding of nickel ionto a fusion polypeptide comprising a polyhistidine tag. Similarly,isolation of a fusion polypeptide comprising a tag can be performed, forexample, by passing a fusion polypeptide comprising a myc epitope over acolumn having an anti-c-myc epitope antibody bound thereto, then elutingthe bound fusion polypeptide, or by passing a fusion polypeptidecomprising a polyhistidine tag over a nickel ion or cobalt ion affinitycolumn and eluting the bound fusion polypeptide. Methods for detectingor isolating such fusion polypeptides will be well known to those in theart, based on the selected detectable label or tag (see, for example,Hopp et al., Bio Technology 6:1204, 1988; U.S. Pat. No. 5,011,912; eachof which is incorporated herein by reference).

A method of the invention also can be used to detectably label anucleotide sequence with a chemical or small organic or inorganic moietysuch that the nucleotide sequence is useful as a probe. For example, ads nucleotide sequence, which has a topoisomerase recognition site, orcleavage product thereof, at a 3′ terminus, can have bound thereto adetectable moiety such as a biotin, which can be detected using avidinor streptavidin, a fluorescent compound (e.g., Cy3, Cy5, Fam,fluorescein, or rhodamine), a radionuclide (e.g., sulfur-35,technicium-99, phosphorus-32, or tritium), a paramagnetic spin label(e.g., carbon-13), a chemiluminescent compound, or the like, such that,upon generating a covalently linked double stranded recombinant nucleicacid molecule according to a method of the invention, the generatednucleic acid molecule will be labeled. Methods of detectably labeling anucleotide sequence with such moieties are well known in the art (see,for example, Hermanson, “Bioconjugate Techniques” (Academic Press 1996),which is incorporated herein by reference). Furthermore, a detectablelabel can be used to allow capture of a ds nucleic acid molecule that isgenerated by the present invention. Finally, a detectable label, forexample biotin, can be used to block ligation of a topoisomerase-chargedend of a first ds nucleotide sequence to a labeled end of a second dsnucleotide sequence, thus providing a method to direct ligation to theunlabelled end of the second ds nucleotide sequence. It should berecognized that such elements as disclosed herein or otherwise known inthe art, including nucleotide sequences encoding cellcompartmentalization domains, or detectable labels or tags, orcomprising transcriptional or translation regulatory elements can beuseful components of a kit as disclosed herein.

A method of the invention provides a means to conveniently generate dsrecombinant nucleic acid molecules that encode chimeric polypeptidesuseful, for example, for performing a two hybrid assay. In such amethod, the first ds nucleotide sequence encodes a polypeptide, or arelevant domain thereof, that is suspected of having or being examinedfor the ability to interact specifically with one or more otherpolypeptides. The first ds nucleotide sequence is modified as disclosedherein to contain a topoisomerase recognition site at one or both endsand, if desired, a 5′ overhanging sequence. The second ds nucleotidesequence, to which the first ds nucleotide sequence is to becovalently-linked according to a method of the invention, can encode atranscription activation domain or a DNA binding domain (Example 2.C),and contains a topoisomerase recognition site, or cleavage productthereof, and a 5′ overhanging sequence complementary to that at the endof the first ds nucleotide sequence to which it is to be linked. Uponcontact with a topoisomerase, if the nucleotide sequences are notalready topoisomerase-charged, a first hybrid useful for performing atwo hybrid assay (see, for example, Fields and Song, Nature 340:245-246,1989; U.S. Pat. No. 5,283,173; Fearon et al., Proc. Natl. Acad. Sci.,USA 89:7958-7962, 1992; Chien et al., Proc. Natl. Acad. Sci., USA88:9578-9582, 1991; Young, Biol. Reprod. 58:302-311(1998), each of whichis incorporated herein by reference), or modified form of a two hybridassay such as the reverse two hybrid assay (Leanna and Hannink, Nucl.Acids Res. 24:3341-3347, 1996, which is incorporated herein byreference), the repressed transactivator system (U.S. Pat. No.5,885,779, which is incorporated herein by reference), the proteinrecruitment system (U.S. Pat. No. 5,776,689, which is incorporatedherein by reference), and the like, is generated. Similar methods areused to generate the second hybrid protein, which can comprise aplurality of polypeptides to be tested for the ability to interact withthe polypeptide, or domain thereof, of the first hybrid protein.

Similarly, such a method of generating a chimeric protein can beperformed according to a method of the current invention for generatinga ds recombinant nucleic acid molecule covalently linked in one strand,using first and second ds nucleotide sequences comprising asite-specific topoisomerase recognition site (e.g., a type IA or a typeII topoisomerase recognition site), or cleavage product thereof, atleast at one 5′ terminus of an end to be joined, wherein the dsnucleotide sequences can further comprise complementary 3′ overhangsupon cleavage by the topoisomerase.

Similarly, such a method of generating a chimeric protein can beperformed according to a method of the current invention for generatinga ds recombinant nucleic acid molecule covalently linked in both strandsusing first and second ds nucleotide sequences comprising atopoisomerase recognition site, or cleavage product thereof, at least atthe 5′ terminus of the ends to be joined, wherein the ds nucleotidesequences can further comprise complementary 3′ overhangs upon cleavageby the topoisomerase; or one of the first or second ds nucleotidesequences can comprise topoisomerase recognition sites, or cleavageproducts thereof, at the 5′ terminus and the 3′ terminus of at least oneend, and the other ds nucleotide sequence can contain a 3′ hydroxylgroup and a 5′ hydroxyl group at the end to be joined, and wherein, uponcleavage by the topoisomerases, the topoisomerase-charged ds nucleotidesequence can contain a 5′ or 3′ overhang that is complementary to, andfacilitates hybridization to, a 5′ or 3′ overhang, respectively, at theend of the other ds nucleotide sequence to be joined.

As disclosed herein, a first ds nucleotide sequence can be one of aplurality of nucleotide sequences, for example, a cDNA library, acombinatorial library of nucleotide sequences, or a population ofvariegated nucleotide sequences. As such, a particularly usefulembodiment of a method of the invention is in generating recombinantpolynucleotides encoding chimeric polypeptides for performing a highthroughput two hybrid assay for identifying protein-protein interactionsthat occur among populations of polypeptides (see U.S. Pat. No.6,057,101 and U.S. Pat. No. 6,083,693, each of which is incorporatedherein by reference). In such a method, two populations (pluralities) ofnucleotide sequences encoding polypeptides are examined, each pluralityhaving a complexity of from a few related but different nucleotidesequences to as high as tens of thousands of such sequences. Byperforming a method of the invention, for example, using a PCR primerpair to amplify each nucleotide sequence in the plurality, wherein atleast one primer of the PCR primer pair comprises at least atopoisomerase recognition site or complement thereof, covalently linkedrecombinant polynucleotides encoding a population of chimeric baitpolypeptides and a population of chimeric prey polypeptides readily canbe generated by contacting the amplified pluralities of nucleotidesequences, each of which comprises a topoisomerase recognition site,with a topoisomerase and a nucleotide sequence, which contains atopoisomerase recognition site and encodes a transcription activationdomain or a DNA binding domain.

In practicing a method of the invention, a first ds nucleotide sequencealso can encode a ribonucleic acid (RNA) molecule, which can function,for example, as a riboprobe, an antisense nucleotide sequence, aribozyme, a triplexing nucleotide sequence, interference RNA (RNAi), ora suppressor tRNA, or can be used in an in vitro translation reaction,and the second ds nucleotide sequence can encode a regulatory elementuseful for expressing an RNA from the first nucleotide sequence (seeExample 2.A). For example, where it is desired to produce a large amountof RNA, a second ds nucleotide sequence component for performing amethod of the invention can comprise an RNA polymerase promoter such asa T7, T3 or SP6 RNA polymerase promoter. Where the RNA molecule is to beexpressed in a cell, for example, an antisense molecule to be expressedin a mammalian cell, the second (or other) ds nucleotide sequence caninclude a promoter that is active in a mammalian cell, particularly atissue specific promoter, which is active only in a target cell.Furthermore, where the RNA molecule is to be translated, for example, ina coupled in vitro transcription/translation reaction, the firstnucleotide sequence or second (or other) nucleotide sequence can containappropriate translational regulatory elements (see Example 2.B).

The methods of the invention can be used, for example, to generatecovalently linked recombinant nucleic acid molecules that encodesuppressor tRNA molecules. The nucleotide sequence encoding thesuppressor tRNA can be operatively linked to an expression controlelement, particularly a transcriptional promoter, which can beconstitutively active or inducible, and can be operative in prokaryoticcells or eukaryotic cells. In addition, the same recombinant nucleicacid molecule or a different recombinant nucleic acid molecule cancontain a first and second coding sequence, which are separated by anucleotide sequence containing a STOP codon that can be suppressed bythe suppressor tRNA. Expression of the suppressor tRNA can then suppressthe STOP codon, thereby allowing the generation of fusion protein. Forexample, where the suppressor tRNA is expressible from an induciblepromoter, the system, which can be introduced into a cell, provides ameans to express a polypeptide encoded by the first coding sequence (inthe absence of expression of the suppressor tRNA) or a fusion proteincomprising the polypeptide encoded by the first coding sequenceoperatively linked to the polypeptide encoded by the second codingsequence (in the presence of expression of the suppressor tRNA), asdesired, simply by including or excluding the inducing agent specificfor the inducible promoter. The polypeptides of such a system can be anypolypeptide as exemplified herein or otherwise known in the art.

Methods of the invention may also be used to produce constructs whichallow for silencing of genes in vivo. One method of silencing genesinvolves the production of double stranded RNAi (see, for example, Metteet al., EMBO J. 19:5194-5201, 2000, which is incorporated herein byreference). The mechanism by which RNAi is believed to function, whichis reviewed in Fjose et al., Biotechnol. Ann. Rev. 7:31-57, 2001,appears to be based on the ability of double stranded RNA to induce thedegradation of specific RNA molecules. This mechanism is reported toinvolve the conversion of double-stranded RNA into short RNAs thatdirect ribonucleases to homologous RNA targets (e.g., mRNA targets).Methods of the invention can be used in a number of ways to producemolecules such as RNAi. Thus, expression products of nucleic acidmolecules of the invention can be used to silence gene expression.

One example of a nucleic acid molecule designed to produce RNAi is amolecule in which a nucleic acid segment is linked to one or morepromoters such that RNA corresponding to both strands are produced astwo separate transcripts or as part of the same transcript. For example,two separate RNA polymerase promoters, which can be the same ordifferent (e.g., a T7 promoter and/or an SP6 promoter) can be located 5′and 3′ to a polynucleotide sequence encoding a polypeptide. Further, theRNA polymerase promoters can be operatively linked to the expressiblepolynucleotide such that transcription driven by each promoter resultsin the production of RNA corresponding to each strand of the expressiblepolynucleotide. Thus, transcription from one promoter results in theproduction of a sense RNA and transcription from the other promoterresults in the production of an antisense RNA. Since the RNA strands arecomplementary, they can hybridize to each other under physiologicalconditions to produce an RNAi molecule.

Another example of a recombinant nucleic acid molecule that can be usedto produce RNAi is one in which an open reading frame is flanked on eachend by promoters that drive transcription of the open reading frame inopposing directions. As a third example, double stranded RNA can beproduced from a recombinant nucleic acid molecule encoding an RNAmolecule having a “snapback” region (e.g., a region that is six, seven,eight, nine ten, etc. nucleotides in length) at one terminus. Such anRNA transcript can form a hairpin turn at or near one terminus and, whenincubated under appropriate conditions in the presence of an RNAdependent RNA polymerase, the double stranded region formed by thehairpin can prime second strand synthesis to form a double stranded RNAmolecule such as an RNAi molecule.

Nucleotide sequence designed to produce RNAi from a recombinant nucleicacid molecules as described above can, but need not, correspond to theentire coding sequence of a gene (i.e., at least the portion containingall of the exons) or a full length open reading frame (ORF). Forexample, when the nucleotide sequence corresponds to a portion of an ORFand, therefore, encodes an RNA molecule that does not correspond to allof the ORF, the nucleotide sequence can include at least about 15 (e.g.,about 20, about 30, about 40, about 50, about 60, etc.) nucleotides, forexample, at least about 15 to about 30 (e.g., 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides at the 5′ end ofthe ORF, the 3′ end of the ORF, or internal to the ORF. Thus, inparticular embodiments, the invention provides methods for preparingrecombinant nucleic acid molecules containing at least three covalentlyoperatively linked nucleotide sequences. In some embodiments, at leasttwo of the nucleotide sequences share at least one region of sequenceidentity (e.g., a region at least about 20, at least about 30, at leastabout 40, at least about 50, at least about 60, at least about 70, atleast about 80, at least about 90, at least about 100 nucleotides, etc.)nucleotides in length, for example, a region of about 15 to 30 (e.g.,15, 16, 17, 18, 19, 20, 21, 22,23,24,25, 26,27,28,29, or 30) nucleotidesin length. In other embodiments, one nucleotide sequence is flanked by aregion that can confer transcription from the interior portion of thenucleotide sequence molecule in opposing directions, thus allowing thegeneration of sense and antisense RNA transcripts. As such, theinvention provides covalently linked recombinant nucleic acid moleculesprepared by methods of the invention, and further provides methods ofusing of such molecules to either inhibit gene expression or facilitatedegradation of specific target RNA molecules.

The invention also provides methods for generating covalently linkedrecombinant nucleic acid molecules that can be used to express antisenseRNA (e.g., antisense mRNA). Methods similar to those described above forthe production of RNAi can be employed, although only the non-codingstrand generally will be transcribed, thereby generating antisense RNAmolecules.

Gene silencing methods involving the use of compounds such as RNAi andantisense RNA, for example, are particularly useful for identifying genefunctions. More specifically, gene silencing methods can be used toreduce or inhibit the expression of one or more genes in a cell ororganism. Phenotypic manifestations associated with the selectiveinhibition of gene functions can then be used to assign role to the“silenced” gene or genes. As an example, Chuang et al. (Proc. Natl.Acad. Sci., USA 97:4985-4990, 2000) demonstrated that in vivo productionof RNAi can alter gene activity in Arabidopsis thaliana. Thus, theinvention provides methods for regulating expression of nucleic acidmolecules in vivo (e.g., in cells and tissues) and/or in vitro byexpressing RNAi molecules, antisense RNA molecules, or a combinationthereof. The invention further provides methods for preparing covalentlylinked recombinant nucleic acid molecules useful for producing RNA thatcorresponding to one or both strands of an expressible polynucleotide.

In related embodiments, promoters that drive transcription of a senseRNA or antisense RNA can be either constitutive (e.g., CMV promoter,SV40 promoter, etc.), inducible (e.g., a metallothionein promoter,etc.), or repressible. Thus, for example, two different induciblepromoters can be used to drive transcription of sense RNA and antisenseRNA. In such an instance, promoter activation can be used to induceproduction of sense RNA, antisense RNA, or both sense RNA and antisenseRNA. Further, the amount of sense RNA and/or antisense RNA produced canbe related by using, for example, graduated induction and/orderepression of the promoters.

The invention also relates to methods of generating a covalently linkedrecombinant nucleic acid molecule encoding a ribozyme, as well as tocompositions containing such recombinant nucleic acid molecules andmethods of using such molecules for gene silencing. In particular, theinvention provides antisense RNA/ribozymes fusions, which comprise 1)antisense RNA corresponding to a target gene and 2) one or moreribozymes that cleave RNA (e.g., hammerhead ribozyme, hairpin ribozyme,delta ribozyme, Tetrahymena L-21 ribozyme, etc.). Further provided bythe invention are vectors that express such fusions, methods forproducing such vectors, and methods for using such vector to suppressgene expression.

Expression of antisense molecules fused to ribozymes can be used, forexample, to cleave specific RNA molecules in a cell because theantisense RNA portion of the transcript can be designed to hybridize toparticular mRNA molecules. Further, the ribozyme portion of thetranscript can be designed to cleave the RNA molecule to which it hashybridized. For example, the ribozyme can be one which cleaves doublestranded RNA (e.g., a Tetrahymena L-21 ribozyme).

The present invention further provides nucleotide sequences suitable forperforming cloning reactions in which a first nucleotide, which sharesone or more regions of homology with a second nucleotide sequence, isused to insert all or a portion of the second nucleotide sequence intothe first nucleotide sequence. The invention further providescompositions and methods for performing such cloning reactions.

One example of such a process is RecE/T cloning (see Internatl. Publ.No. WO 01/04288, which is incorporated herein by reference). Typically,in RecE/T cloning, a linear first nucleotide sequence (e.g., a vector)is introduced into a cell that contains 1) regions at the termini thatshare homology with two separate nearby regions (e.g., regions that areabout 20 to 30, or about 20 to 40, or about 20 to 50, or about 30 to 40,or about 40 to 50, or about 40 to 60, or about 40 to 80, or about 50 to90, etc. nucleotides in length) of a second nucleotide sequence, whichis present in the cell (e.g., a plasmid, a bacterial artificialchromosome, a natural chromosome, etc.), 2) a selection marker, and 3)an origin of replication. The linear first nucleotide sequence generallyreplicates only if it becomes circularized. Further, the firstnucleotide sequence typically becomes circularized upon undergoingrecombination with the second nucleotide sequence and acquiring aportion of the second nucleotide sequence, which is intervening betweenthe regions of homology. In such embodiments, the regions of homology inthe first nucleotide sequence will typically be in a reverse orientationas compared to the second nucleotide sequence. Generally, the cell inwhich recombination occurs is one that expresses a recombinase such asRecE/T or RedAlpha/Beta. Thus, the invention provides, in part, methodsfor performing RecE/T cloning, covalently linked ds recombinant nucleicacid molecules prepared by such methods, compositions comprising suchrecombinant nucleic acid molecules, and methods for using such nucleicacid molecules and compositions.

Modifications of the RecE/T process can be used to generate a number ofdifferent end products. For example, when the regions of homology arearranged in various ways, the first nucleotide sequence can be designedto 1) insert into the second nucleotide sequence, or 2) delete a portionof the second nucleotide sequence. Typically, when insertion of thesecond nucleotide sequence into the second nucleotide sequence isdesired, the regions of homology of the first nucleotide sequence are inthe same orientation with respect to the regions of homology in thesecond nucleotide sequence. Further, when deletion of nucleic acid fromthe second nucleotide sequence is desired, the regions of homology ofthe first nucleotide sequence generally are in an inverse orientationwith respect to the regions of homology in the second nucleotidesequence. Also, when insertion of the first nucleotide sequence into thesecond nucleotide sequence is desired, typically the first nucleotidesequence lacks an origin of replication. Accordingly, the presentinvention provides methods for performing such processes, as well asnucleotide sequences and compositions for use in the above methods.

A method of the invention can be particularly useful for generating anexpressible ds recombinant nucleic acid molecule that can be inserted ina site specific manner into a target DNA sequence. The target DNAsequence can be any DNA sequence, particularly a genomic DNA sequence,and preferably a gene for which some or all of the nucleotide sequenceis known. The method can be performed utilizing a first ds nucleotidesequence, which has a first end and a second end and encodes apolypeptide, for example, a selectable marker, wherein the first dsnucleotide sequence comprises a topoisomerase recognition site orcleavage product thereof at the 3′ terminus of each end and, optionally,a hydroxyl group at the 5′ terminus of each end, and wherein,preferably, the 5′ termini comprise 5′ overhanging sequences, which aredifferent from each other; and covalently linking the first dsnucleotide sequence to first and second PCR amplification productsaccording to a method of the invention. The first and secondamplification products are generated from sequences upstream anddownstream of the site at which the construct is to be inserted, andeach amplification product contains a topoisomerase recognition siteand, preferably, a 5′ overhanging sequence, which is generated followingcontact with the site specific topoisomerase. Preferably, the first andsecond amplification products have different 5′ overhanging sequencessuch that each can be linked to a predetermined end of the first dsnucleotide sequence. Such a method similarly can be performed using a dsamplification product comprising a topoisomerase recognition site, orcleavage product thereof, at the 5′ terminus of one or both ends,wherein, upon cleavage by the topoisomerase, the topoisomerase-chargedmolecule can comprise a 3′ overhang at one or both ends containing thetopoisomerase. In addition, the method can be performed using a dsamplification product comprising topoisomerase recognition sites, orcleavage products thereof, at the 5′ terminus and the 3′ terminus of oneor both ends, wherein, upon cleavage by the topoisomerases, thetopoisomerase-charged ds nucleotide sequence preferably contains a 5′ or3′ overhang at one or both ends containing the topoisomerases.

The first and second amplification products are generated using two setsof PCR primer pairs. The two sets of PCR primer pairs are selected suchthat, in the presence of an appropriate polymerase such as Taqpolymerase and a template comprising the sequences to be amplified, theprimers amplify portions of a target DNA sequence that are upstream ofand adjacent to, and downstream of and adjacent to, the site forinsertion of the selectable marker. In addition, the sets of PCR primerpairs are designed such that the amplification products contain atopoisomerase recognition site and, following cleavage by the sitespecific topoisomerase, a 5′ overhanging sequence at the end to becovalently linked to the selectable marker. As such, the first PCRprimer pair includes 1) a first primer, which comprises, in anorientation from 5′ to 3′, a nucleotide sequence complementary to a 5′overhanging sequence of the end of the selectable marker to which theamplification product is to be covalently linked, a nucleotide sequencecomplementary to a topoisomerase recognition site, and a nucleotidesequence complementary to a 3′ sequence of a target DNA sequenceupstream of the insertion site; and 2) a second primer, which comprisesa nucleotide sequence of the target genomic DNA upstream of the 3′sequence to which the first primer is complementary, i.e., downstream ofthe insertion site. The second PCR primer pair includes 1) a firstprimer, which comprises, from 5′ to 3′, a nucleotide sequencecomplementary to the 5′ overhanging sequence of the end of theselectable marker to which it is to be covalently linked, a nucleotidesequence complementary to a topoisomerase recognition site, and anucleotide sequence of a 5′ sequence of a target DNA sequence, whereinthe 5′ sequence of the target genomic DNA is downstream of the 3′sequence of the target DNA sequence to which the first primer of thefirst PCR primer pair is complementary; and the second primer of thesecond primer pair comprises a nucleotide sequence complementary to a 3′sequence of the target DNA sequence that is downstream of the 5′sequence of the target genomic DNA contained in the first primer. Theskilled artisan will recognize that the sequences of the primer that arecomplementary to the target genomic DNA are selected based on thesequence of the target DNA.

Upon contact of the ds nucleotide sequence comprising the selectablemarker, the first and second amplification products, and a topoisomerase(if the molecules are not topoisomerase-charged), a ds recombinantnucleic acid molecule covalently linked in both strands is generatedaccording to a method of the invention. The generated ds recombinantnucleic acid molecule can be further amplified, if desired, using PCRprimers that are specific for an upstream and downstream sequence of thetarget genomic DNA, thus ensuring that only functional constructs areamplified. The generated ds recombinant nucleic acid molecule is usefulfor performing homologous recombination in a genome, for example, toknock-out the function of a gene in a cell, or to confer a novelphenotype on the cell containing the generated recombinant nucleic acidmolecule. The method can further be used to produce a transgenicnon-human organism having the generated ds recombinant nucleic acidmolecule stably maintained in its genome.

A method of the invention also is useful for covalently linking, anadapter or linker sequence to one or both ends of a ds nucleotidesequence of interest, including to each of a plurality of ds nucleotidesequences. For example, where it is desired to put linkers on both endsof a first ds nucleotide sequence, the method can be performed bycontacting a topoisomerase with a first ds nucleotide sequence, whichhas a topoisomerase recognition site, or cleavage product thereof, atone or both 3′ or 5′ termini and which can include hydroxyl groups atboth 5′ termini; and a second ds nucleotide sequence and at least athird double stranded nucleotide sequence, each of which can include atopoisomerase recognition site, or cleavage product thereof at theappropriate 3′ or 5′ terminus and which can also include, wheredesirable, a 5′ hydroxyl group at the same terminus. An appropriateterminus is the terminus to which the linker is to be covalently linkedin at least one strand to the first nucleotide sequence. In oneembodiment, one or both linker sequences contain an overhanging sequencethat is complementary to a sequence at the 5′ terminus of the end of thefirst ds nucleotide sequence to which the linker is to be covalentlylinked, thereby facilitating the initial association of the nucleotidesequences in the proper (predetermined) orientation (see, for example,FIG. 2 and Example 1.B). In performing such a method, the linkersequences comprising the second and at least third nucleotide sequencecan be the same or different.

FIG. 7 shows one example of a process for preparing a ds nucleotidesequence containing a topoisomerase (e.g., a type IA topoisomerase)bound to the 5′ terminus of one end of the sequence, and wherein thesame end further comprise a 3′ overhang (see (4) in FIG. 7). In step A,a nucleotide sequence to be modified with topoisomerase is digested witha restriction enzyme that generates a “sticky” end. The restrictednucleotide sequence is then contacted in step B with a linear, singlestranded nucleotide sequence which contains a topoisomerase attached the5′ terminus and a ligase (e.g., a DNA ligase such as T4 DNA ligase). Thelinear, single stranded nucleotide sequence also contains a region atthe 3′ terminus which shares sufficient sequence complementarity to the“sticky” end generated by the restriction enzyme, such that the twomolecules will hybridize. Thus, in step B, the two nucleotide sequencesare ligated to each other. In step C, the product of the second step iscontacted with a third nucleotide sequence which shares sequencecomplementarity to portions of the linear, single stranded nucleic acidmolecule generated in step B, and a ligase. The product of step C, shownin (4), is a ds nucleotide sequence containing a topoisomerase attachedto the 5′ terminus of one end and a 3′ overhang on the same end. It willbe recognized that numerous variations of the exemplified method arewithin the scope of the invention. For example, similar processes can beperformed to prepare nucleic acid molecules which comprise topoisomeraseattached to the 3′ terminus of one end or which have a 5′ overhang orare blunt ended at the end to which a topoisomerase is attached. Inanother example, the nucleotide sequence labeled number 3 in FIG. 7 canbe produced in the following manner: a ds nucleotide sequence can bedigested with a restriction enzyme to generate a ds nucleotide sequencewith a single-stranded 5′ overhang that includes a type IA topoisomeraserecognition site. The ds nucleotide sequence with the single strandedoverhang can then be contacted with type IA topoisomerase to generate atype IA topoisomerase-charged ds nucleotide sequence.

FIG. 8 shows two embodiments of the invention in which single strandedor double stranded DNA is covalently linked to single stranded RNA.Where single stranded DNA is joined to single stranded RNA, the 3′ endof the ribonucleotide sequence is covalently linked to the 5′ end of thedeoxyribonucleotide sequence. Where double stranded DNA is joined tosingle stranded RNA, the 3′ terminus of the ribonucleotide sequenceshares sufficient sequence complementarity to the 3′ overhang of thedeoxyribonucleotide sequence such that the two molecules hybridize. Asabove, the 3′ end of the ribonucleotide sequence is also covalentlylinked to the 5′ end of the deoxyribonucleotide sequence. As will berecognized, numerous variations of the above are within the scope of theinvention. For example, the RNA molecule can be double stranded. Inanother example, all of the nucleotide sequences can bedeoxyribonucleotide sequences.

The present invention provides a ds recombinant nucleic acid moleculehaving, or which can be made to have, a first end and a second end, eachend including a 5′ terminus and a 3′ terminus, wherein the vectorcomprises a site-specific type IA topoisomerase recognition site at ornear a 5′ terminus of the first end, the second end, or both the firstend and the second end. The ds recombinant nucleic acid molecule canfurther include a type IB topoisomerase recognition site at or near a 3′termini of an end that does not include a type IA topoisomeraserecognition site. The ds recombinant nucleic acid molecule can be avector.

The present invention further provides a topoisomerase-charged dsrecombinant nucleic acid molecule having a first end and a second end,each end having a 5′ terminus and a 3′ terminus, wherein a site-specifictype IA topoisomerase is bound at the 5′ terminus of the first end, thesecond end, or both the first end and the second end. For example, thetopoisomerase-charged ds recombinant nucleic acid molecule can include atype IA topoisomerase bound at the 5′ termini of each of the first andsecond ends. The topoisomerase-charged nucleic acid ds recombinantnucleic acid molecule can include a type IB topoisomerase bound at a 3′termini of an end not bound by a type IA topoisomerase. Thetopoisomerase-charged ds recombinant nucleic acid molecule can be avector.

The present invention also provides kits, which contain componentsuseful for conveniently practicing the methods of the invention. Kits ofthe invention can contain any number of components, and generallycontain at least two components. For example, a kit of the invention cancontain 1) a first nucleotide sequence containing one or moretopoisomerase recognition sites, and 2) instructions for covalentlylinking the first nucleotide sequence to a second (or other) nucleotidesequence using a method as disclosed herein. In particular embodiments,the instructions provide methods for covalently linking two or morenucleotide sequences in one or both strands. For example, theinstructions can be for covalently linking two or more ds nucleotidesequences in both strands, and can include instructions for obtaining asecond (or other) ds nucleotide sequence that contains a topoisomeraserecognition site or that is topoisomerase-charged on one or more terminithat are to covalently linked to the first ds nucleotide sequence, orcan include instructions for making or obtaining a primer, which can beone of a primer pair, that includes, for example, a nucleotide sequencecomplementary to a type IB topoisomerase recognition site, such that aterminus of an amplification product generated using such a primer pair(including such a primer) can be covalently linked (in the presence of atype IB topoisomerase) to an end of a first ds nucleotide sequence thathas a type IB topoisomerase recognition site at 3′ terminus of the endto be linked or that is topoisomerase-charged at that terminus. In arelated embodiment, the first nucleotide sequence is topoisomeraseadapted (topoisomerase-charged) prior to inclusion in the kit.

In one embodiment, a kit of the invention contains a first ds nucleotidesequence, which encodes a polypeptide, particularly a selectable marker,and contains a topoisomerase recognition site at each end. Preferably,the first nucleotide sequence comprises a topoisomerase-activatednucleotide sequence. More preferably, the topoisomerase-charged firstnucleotide sequence comprises a 5′ overhanging sequence at each end, andmost preferably the 5′ overhanging sequences are different from eachother. Optionally, each of the 5′ termini comprises a 5′ hydroxyl group.

In addition, the kit can contain at least a nucleotide sequence (orcomplement thereof) comprising a regulatory element, which can be anupstream or downstream regulatory element, or other element, and whichcontains a topoisomerase recognition site at one or both ends.Preferably, the kit contains a plurality of ds nucleotide sequences,each comprising a different regulatory element or other element, forexample, a sequence encoding a tag or other detectable molecule or acell compartmentalization domain. The different elements can bedifferent types of a particular regulatory element, for example,constitutive promoters, inducible promoters and tissue specificpromoters, or can be different types of elements including, for example,transcriptional and translational regulatory elements, epitope tags, andthe like. Such ds nucleotide sequences can be topoisomerase-activated,and can contain 5′ overhangs or 3′ overhangs that facilitate operativelycovalently linking the elements in a predetermined orientation,particularly such that a polypeptide such as a selectable marker isexpressible in vitro or in one or more cell types.

The kit also can contain primers, including first and second primers,such that a primer pair comprising a first and second primer can beselected and used to amplify a desired ds recombinant nucleic acidmolecule covalently linked in one or both strands, generated usingcomponents of the kit. For example, the primers can include firstprimers that are complementary to elements that generally are positionedat the 5′ end of a generated ds recombinant nucleic acid molecule, forexample, a portion of a ds nucleotide sequence comprising a promoterelement, and second primers that are complementary to elements thatgenerally are positioned at the 3′ end of a generated ds recombinantnucleic acid molecule, for example, a portion of a ds nucleotidesequence comprising a transcription termination site or encoding anepitope tag. Depending on the elements selected from the kit forgenerating a ds recombinant nucleic acid molecule covalently linked inboth strands, the appropriate first and second primers can be selectedand used to amplify a full length functional construct.

In another embodiment, a kit of the invention contains a plurality ofdifferent elements, each of which can be topoisomerase-activated at oneor both ends, and each of which can contain a 5′ overhanging sequence ora 3′overhanging sequence or a combination thereof. The 5′ or 3′overhanging sequences can be unique to a particular element, or can becommon to plurality of related elements, for example, to a plurality ofdifferent promoter element. Preferably, the 5′ overhanging sequences ofelements are designed such that one or more elements can be operativelycovalently linked to provide a useful function, for example, an elementcomprising a Kozak sequence and an element comprising a translationstart site can have complementary 5′ overhangs such that the elementscan be operatively covalently linked according to a method of theinvention.

The plurality of elements in the kit can comprise any elements,including transcription or translation regulatory elements; elementsrequired for replication of a nucleotide sequence in a bacterial,insect, yeast, or mammalian host cell; elements comprising recognitionsequences for site specific nucleic acid binding proteins such asrestriction endonucleases or recombinases; elements encoding expressibleproducts such as epitope tags or drug resistance genes; and the like. Assuch, a kit of the invention provides a convenient source of differentelements that can be selected depending, for example, on the particularcells that a construct generated according to a method of the inventionis to be introduced into or expressed in. The kit also can contain PCRprimers, including first and second primers, which can be combined asdescribed above to amplify a ds recombinant nucleic acid moleculecovalently linked in one or both strands, generated using the elementsof the kit. Optionally, the kit further contains a site specifictopoisomerase in an amount useful for covalently linking in at least onestrand, a first ds nucleotide sequence comprising a topoisomeraserecognition site to a second (or other) ds nucleotide sequence, whichcan optionally be topoisomerase-activated ds nucleotide sequences ornucleotide sequences that comprise a topoisomerase recognition site.

In still another embodiment, a kit of the invention contains a first dsnucleotide sequence, which encodes a selectable marker, and contains atopoisomerase recognition site at each end; a first and second PCRprimer pair, which can produce a first and second amplification productsthat can be covalently linked in one or both strands, to the first dsnucleotide sequence in a predetermined orientation according to a methodof the invention. Such a generated construct can be introduced into acell and can incorporate into the genome of the cell by homologousrecombination in a site specific manner, where it can be stablymaintained and can express a heterologous polypeptide in the cell or canknock-out a target gene function. A target gene to be knocked-out, forexample, can be any gene for which at least part of the sequence isknown or can be readily determined and the function of which it isdesired to disrupt, for example, an oncogene, a gene involved inapoptosis, a gene encoding a serine/threonine or a tyrosine kinase, orany other gene.

The first PCR primer pair in a kit of the invention useful forgenerating a ds recombinant nucleic acid molecule covalently linked inboth strands, includes a first primer that comprises, in an orientationfrom 5′ to 3′, a nucleotide sequence complementary to a 5′ overhangingsequence of a ds nucleotide sequence to which it is to be covalentlylinked (for example, an end of the ds nucleotide sequence encoding theselectable marker), a nucleotide sequence complementary to atopoisomerase recognition site, and a nucleotide sequence complementaryto a 3′ sequence of the target DNA sequence. The first PCR primer pairalso includes a second primer that comprises a nucleotide sequence ofthe target DNA sequence upstream of the 3′ sequence to which the firstprimer is complementary.

The second PCR primer pair of a kit useful for generating a dsrecombinant nucleic acid molecule covalently linked in both strands,includes a first primer that comprises, from 5′ to 3′, a nucleotidesequence complementary to a 5′overhanging sequence of a ds nucleotidesequence to which it is to be covalently linked, a nucleotide sequencecomplementary to a topoisomerase recognition site, and a nucleotidesequence of a 5′ sequence of the target DNA sequence, wherein the 5′sequence of the target gene is downstream of the 3′ sequence of thetarget DNA sequence to which the first primer of the first primer pairis complementary. The second PCR primer pair also includes a secondprimer that comprises a nucleotide sequence complementary to a 3′sequence of the target gene that is downstream of the 5′ sequence of thetarget DNA sequence contained in the first primer.

In another embodiment, a kit of the invention useful for generating a dsrecombinant nucleic acid molecule covalently linked in both strandscontains a first ds nucleotide sequence, which encodes a transcriptionactivation domain and comprises a topoisomerase recognition site, orcleavage product thereof, at a 3′ terminus; and a second ds nucleotidesequence, which encodes a DNA binding domain and comprises atopoisomerase recognition site, or cleavage product thereof, at a 3′terminus. Upon cleavage by the site specific topoisomerase, the first orsecond ds nucleotide sequence can have a 5′ overhang, or both sequencescan have 5′ overhangs, which are the same or are different from eachother. Where the ds nucleotide sequences have a 5′ overhang, theoverhang generally is complementary to a ds nucleotide sequence to whichfirst or second ds nucleotide sequence is to be covalently linkedaccording to a method of the invention. The kit also can contain one ora pair of adapters, linkers or the like, which can comprise atopoisomerase recognition site, or cleavage product thereof, at one orboth 3′ termini, and, optionally, a hydroxyl group at the sameterminus/termini. Such adapters, linkers, or the like are selected suchthat they contain a 5′ overhang that is complementary to one or theother of the two ds nucleotide sequences described above and part of thekit.

Similarly, a kit of the invention can contain one or a pair of adapters,linkers or the like, which comprise a topoisomerase recognition site, orcleavage product thereof, at one or both 5′ termini, and, optionally, ahydroxyl group at the same terminus (or termini). Such adapters,linkers, or the like are selected such that they contain a 3′ overhangthat is complementary to one or the other of the two ds nucleotidesequences described above and part of the kit. In addition, the kit cancontain one or a pair of adapters, linkers or the like, which comprise atopoisomerase recognition site, or cleavage product thereof, at one orboth 5′ and/or 3′ termini, and, optionally, a hydroxyl group at the sameterminus/termini.

Adapters, linkers, or the like generally are selected such that theycontain a 5′ and/or a 3′ overhang that is complementary to one or theother of the two ds nucleotide sequences as disclosed herein and part ofthe kit. Such adapters, linkers, or the like can be joined to the endsof ds nucleotide sequences that are to covalently linked to one or theother of the first or second ds nucleotide sequences provided with thekit, thus facilitating the construction of chimeric polynucleotidesencoding the bait and prey polypeptides useful in a two hybrid assay.Such a kit also can contain a PCR primer or primer pair, which can beused to prepare an amplified plurality of nucleotide sequencescomprising a topoisomerase recognition site, or cleavage product thereof(see Table 1 and Example 1).

A PCR primer pair in a kit of the invention, which can be used forgenerating a ds recombinant nucleic acid molecule covalently linked inone strand, can include a first primer that comprises, in an orientationfrom 5′ to 3′, a nucleotide sequence of a 5′ overhanging sequence of ads nucleotide sequence to which it is to be linked (for example, an endof the ds nucleotide sequence encoding the selectable marker), atopoisomerase recognition site (e.g., a type IA or type II topoisomeraserecognition site), and a nucleotide sequence complementary to a 5′sequence of the target DNA sequence. The PCR primer pair also includes asecond primer that comprises a nucleotide sequence of the target DNAsequence downstream of the 5′ sequence to which the first primer iscomplementary.

In another embodiment, a kit of the invention contains a first dsnucleotide sequence, which encodes a transcription activation domain andcomprises a site-specific topoisomerase recognition site (e.g., a typeIA or a type II topoisomerase recognition site), or cleavage productthereof, at a 5′ terminus; and a second ds nucleotide sequence, whichencodes a DNA binding domain and comprises a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite), or cleavage product thereof, at a 5′ terminus. Upon cleavage bythe site specific topoisomerase, the first or second ds nucleotidesequence can have a 3′ overhang, or both sequences can have 3′overhangs, which are the same or are different from each other. Wherethe ds nucleotide sequences have a 3′ overhang, the overhang generallyis complementary to a ds nucleotide sequence to which first or second dsnucleotide sequence is to be linked according to a method of theinvention. The kit also can contain one or a pair of adapters, linkersor the like, which comprise a site-specific topoisomerase recognitionsite (e.g., a type IA or a type II topoisomerase recognition site), orcleavage product thereof, at one or both 5′ termini, and which cancontain a 5′ overhang that is complementary to one or the other of thetwo ds nucleotide sequences of the kit.

A kit of the invention also can contain a first isolatedtopoisomerase-charged ds nucleotide sequence and at least a secondisolated topoisomerase-charged ds nucleotide sequence, wherein thesequences of the first and at least second ds nucleotide sequences aredifferent from each other; or can contain at least two different dsnucleotide sequences, each of which comprises a topoisomeraserecognition site at or near one or both ends, and a site specifictopoisomerase, which can bind to and cleave the at least two differentds nucleotide sequences at the topoisomerase recognition site; or cancontain a site specific topoisomerase and a covalently linked dsrecombinant nucleic acid molecule, wherein the covalently linked dsrecombinant nucleic acid molecule comprises at least one topoisomeraserecognition site for the site specific topoisomerase in eachcomplementary strand, wherein the topoisomerase recognition sites ineach complementary strand are within about fifty nucleotides of eachother, and wherein the site specific topoisomerase can bind to andcleave the topoisomerase recognition site in each complementary strand.In addition, a kit of the invention can contain a first ds nucleotidesequence, which contains a first end and a second end, and encodes apolypeptide, said first ds nucleotide sequence further comprising atopoisomerase bound at each end; and a plurality of ds nucleotidesequence populations, wherein each ds nucleotide sequence in apopulation contains a first end and a second end, and comprises aregulatory element, each ds nucleotide sequence further comprising atopoisomerase bound at the first end, the second end or both ends,wherein each population in the plurality is different from each otherpopulation, and wherein each ds nucleotide sequence in a populationcontains the same overhanging sequence, which is different from theoverhanging sequence in the ds nucleotide sequences in each otherpopulation. Such a kit also can contain PCR primers specific for the dsnucleotide sequences in each population of nucleotide sequences. In oneembodiment, the polypeptide encoded by the first ds nucleotide sequenceis a selectable marker.

A ds recombinant nucleic acid molecule covalently linked in one or bothstrands, and generated according to a method of the invention, can beused for various purposes, including, for example, for expressing apolypeptide in a cell, for diagnosing or treating a pathologiccondition, or the like. As such, the present invention provides amedicament, which can be useful for treating a pathologic condition byexpressing a polypeptide in one or more cells or by expressing anantisense molecule, or the like. Such a ds recombinant nucleic acidmolecule can be provided to a cell by contacting the cell ex vivo, thenadministering the cell to the subject, such a method also allowing forselection and/or expansion of the cells containing the ds recombinantnucleic acid molecule prior to such administration, or can be provideddirectly to the subject. For administration to a living subject, the dsrecombinant nucleic acid molecule, which is covalently linked in one orboth strands, generally is formulated in a composition suitable foradministration to the subject. Thus, the invention provides compositionscontaining a ds recombinant nucleic acid molecule covalently linked inone or both strands, generated according to a method of the invention.As disclosed herein, such nucleic acid molecules are useful asmedicaments for treating a subject suffering from a pathologicalcondition.

A composition for administration generally is formulated using one ormore pharmaceutically acceptable carriers as well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the composition,which can be, for example, orally or parenterally such as intravenously,and by injection, intubation, or other such method known in the art. Acomposition of the invention also can contain a second reagent such as adiagnostic reagent, nutritional substance, toxin, or therapeutic agent,for example, a cancer chemotherapeutic agent.

The ds recombinant nucleic acid molecule covalently linked in one orboth strands, can be incorporated within an encapsulating material suchas into an oil-in-water emulsion, a microemulsion, micelle, mixedmicelle, liposome, microsphere or other polymer matrix (see, forexample, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. 1984); Fraley, et al., Trends Biochem. Sci., 6:77, 1981,each of which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer. “Stealth” liposomes (see, forexample, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each ofwhich is incorporated herein by reference) are an example of suchencapsulating materials particularly useful for preparing apharmaceutical composition, and other “masked” liposomes similarly canbe used, such liposomes extending the time that a nucleic acid moleculeremains in the circulation. Cationic liposomes, for example, also can bemodified with specific receptors or ligands (Morishita et al., J. Clin.Invest., 91:2580-2585, 1993, which is incorporated herein by reference).The nucleic acid molecule also can be introduced into a cell bycomplexing it with an adenovirus-polylysine complex (see, for example,Michael et al., J. Biol. Chem. 268:6866-6869, 1993, which isincorporated herein by reference). Such compositions can be particularlyuseful for introducing a nucleic acid molecule into a cell in vivo or invitro, including ex vivo, wherein the cell containing the nucleic acidmolecule is administered back to the subject (see U.S. Pat. No.5,399,346, which is incorporated herein by reference). A nucleic acidmolecule generated according to a method of the invention also can beintroduced into a cell using a biolistic method (see, for example, Sykesand Johnston, supra, 1999).

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Construction of Covalently Linked Double Stranded RecombinantNucleic Acid Molecules Using Topoisomerase

This experiment demonstrates that topoisomerase can be used to producecovalently linked double stranded (ds) recombinant nucleic acidmolecules.

A. Methods

Except where indicated, experiments were performed using the followingmethods. PCR was performed in 50 Tl reactions, including 10 ng plasmid(template), 100 ng each primer, 2.5 Units Taq DNA polymerase (Sigma), 5Tl 10×PCR buffer, and 4 Tl of dNTPs (200 TM each). An initialdenaturation was performed by incubating the reaction at 94° C. for 4min; followed by 30 cycles of PCR using 94° C. (45 sec) fordenaturation, 55° C. (45 sec) for primer annealing and 72° C. (1 min perkb of target sequence) for extension. After cycling, the reactions wereincubated at 72° C. (10 min), and then placed at 4° C.

Topoisomerase joining reactions were performed in 5 Tl, including 50-100ng each amplified element (PCR-generated or synthetic), 0.5 Tl 500 mMTris (pH 7.5), and 0.5 Tg topoisomerase. Reactions were incubated atroom temperature for 5 min, then 1-2 Tl of the Topo-linked product wasused for linear fragment generation.

Linear fragment generation by PCR was performed in 50 Tl reactions,including 1-2 Tl of the Topo-linked product (template), 100 ng eachprimer, 2.5 U Taq DNA polymerase (Sigma), 5 Tl 10×PCR buffer, and 4 TldNTPs (200 TM each). PCR was performed as described above.

The resultant linear fragment was purified using a SNAP Miniprep Kit(Invitrogen) as described by the manufacturer. Essentially, 100 Tl PCRproduct was mixed with 300 Tl Binding Buffer; 750 Tl isopropanol, andthe mixture was applied to a SNAP Miniprep Column/Collection Tube andcentrifuged at 7,000 rpm for 30 sec. The column was washed with 700 TlWash Buffer, centrifuged at 7,000 rpm for 30 sec; then washed with 900Tl 1×Final Wash and centrifuged at 7,000 rpm for 30 sec. The column wasthen centrifuged at 7,000 rpm for an additional 30 sec to remove allremaining liquid. Water (30 to 50 Tl) was added and the column wascentrifuged at 7,000 rpm for 30 sec to elute the purified DNA. DNAconcentration was determined by spectrophotometry.

B. Generation of Topoisomerase Linked Linear Nucleic Acid Molecules

PCR primers were designed to examine the directional addition ofelements to the coding sequence of green fluorescent protein (GFP; seeFIG. 2). The CMV promoter (approximately 700 bp) and BGH polyadenylationsignal sequence (approximately 380 bp) were amplified from apCMV/myc/nuc plasmid template, and the GFP element (approximately 700bp) was amplified from a pcDNA3.1/GFP plasmid template (Invitrogen)using the primers indicated in FIG. 2. The resultant amplificationproducts were joined using topoisomerase as described above, and aportion of the ligation reaction was used as template for PCR withprimers F6945 (SEQ ID NO: 11) and F6948 (SEQ ID NO: 15) to amplify theentire construct (CMV+GFP+BGH; approximately 1,700 bp). In addition, 5Tl of the ligation mixture was treated with proteinase K for 30 min at37° C. to remove any bound topoisomerase, and then subjected toelectrophoresis on a 3-8% NuPAGE Tris-acetate gel to examine the ligatedproducts.

Only a small amount of ligation product of the correct size (1.7 kb) wasobserved when the recombinant nucleic acid molecules were generatedusing elements having palindromic overhanging sequence (FIG. 2A or 2B),whereas significant quantities of the desired product were generatedusing elements having non-palindromic overhangs (FIG. 2C). These resultsdemonstrate that the efficiency of generating a ds recombinant nucleicacid molecule covalently linked in both strands containing nucleotidesequences operatively linked in a predetermined orientation is relatedto the nature of the overhang sequence. In particular, the selection ofoverhanging sequences that lack palindromic regions result in theefficient generation of a desired ds recombinant nucleic acid moleculecovalently linked in both strands, whereas the presence of palindromicsequences in the overhangs allows the formation of ligation productsother than the intended product, thus decreasing the efficiency ofgenerating a desired product.

EXAMPLE 2 Functional Characterization of Topoisomerase-Generated DSRecombinant Nucleic Acid Molecules

This example demonstrates that a method of the invention provides ameans to generate functional ds recombinant nucleic acid moleculescovalently linked in both strands.

A. Expression of Sense and Antisense mRNA from a Topo-ligated Construct

The ability to create a ds recombinant nucleic acid molecule containingfunctional upstream and downstream elements flanking a gene of interestwas examined using two synthetic elements containing either a T7 or a T3promoter sequence. The elements were made by annealing pairs ofsynthetic oligonucleotides. The T7 linker was generated by mixing equalmolar amounts of T7top (F9304; SEQ ID NO: 20) and T7bottom (F9305; SEQID NO: 21) oligonucleotides (Table 1). The T3 linker was generated bymixing equal molar amounts of T3top (F9661; SEQ ID NO: 23) and T7bottom(F9662; SEQ ID NO: 24) oligonucleotides (Table 1). The mixtures wereheated in boiling water for 5 min, then allowed to cool to roomtemperature. Both elements were designed to contain a topoisomeraserecognition site at one end.

The GFP gene was amplified with GFP primers F8418 (SEQ ID NO: 17) andF8420 (SEQ ID NO: 18; Table 1; see, also, FIG. 2C). Unpurified GFP PCRproduct (2 Tl) was mixed with 50 ng of T7 linker and 50 ng of T3 linker,topoisomerase was added, and the topo-joining reaction was allowed toproceed at room temperature for 5 min. Two Tl of the joining reactionwas used as template for a 50 Tl PCR reaction with primers for the T7and T3 sequences.

After amplification, a 4 Tl aliquot of the PCR reaction was used astemplate for in vitro transcription. The reaction was performed using aPromega RiboProbe In Vitro Transcription Systems kit according to themanufacturer's instruction. The reaction was allowed to proceed for 60min at 37° C. with T7 or T3 RNA polymerase (final volume, 20 Tl).Aliquots of the in vitro transcription reactions were digested withRNase or DNase, then undigested and digested samples were subjected toelectrophoresis in a 2% TBE gel. A predominant band of the predictedsize (either sense or antisense orientation) was observed in theundigested samples. No decrease in the product band was noted in samplestreated with DNase. The product bands disappeared when samples weretreated with RNase indicating the product was RNA. These resultsdemonstrate that topoisomerase can be used according to a method of theinvention to generate a ds recombinant nucleic acid molecule covalentlylinked in both strands in a predetermined orientation, and that an RNAtranscript can be expressed from such a nucleic acid molecule.

B. Expression of a Translation Product from a Topo-ligated Construct

The ability of topoisomerase ligated polynucleotide to support coupledin vitro transcription/translation was examined. A ds recombinantnucleic acid molecule was generated according to a method of theinvention by linking an element containing a T7 promoter (plus a Kozaksequence) to lacZ PCR products of 1 kb, 2 kb, or 3 kb. Two Tl of thegenerated products were used as template for PCR amplification reactions(primers, SEQ ID NOS: 25-28; Table 1). Unpurified aliquots of theamplification reactions (3 Tl) were used as templates for coupledtranscription/translation with a TNT T7 Quick for PCR DNA Kit accordingto the manufacturer's instructions (Promega).

Two Tl aliquots from each reaction were separated by electrophoresis ona Tris-glycine gel (Novex), then visualized by autoradiography, whichrevealed protein products that migrated at the expected sizes. Theseresults demonstrate that a method of the invention can be used toproduce a ds recombinant nucleic acid molecule covalently linked in bothstrands useful as a template for expressing a polypeptide by a coupledin vitro transcription/translation reaction.

C. Generation of Topo-ligated Constructs for Performing a Two HybridAssay

Two hybrid assays provide a powerful method for detectingprotein-protein interactions in vivo. These assays are based on the factthat many eukaryotic transcriptional activators consist of twophysically and functionally separable domains, including a DNA bindingdomain, which binds to a specific DNA sequence, and a transcriptionalactivation domain, which interacts with the basal transcriptionalmachinery. The association of a transactivation domain with a DNAbinding domain can promote the assembly of a functional RNA polymeraseII complex, thereby allowing transcriptional activation, for example, ofa detectable reporter gene (Field and Song, supra, 1989). Where a firstprotein, X, is fused to a DNA binding domain, for example, a GAL4binding domain, and a second protein, Y, which can be the same ordifferent from X, is fused into a transactivation domain, for example, aVP16 domain, an interaction of proteins X and Y can be identified bydetecting transcription of a reporter gene having a GAL4 promoter.

The ability of a method of the invention to generate linear constructsfor expressing fusion proteins for performing a mammalian two-hybridassay was examined. PCR was used to generate GAL4 (F10779 and F12667primers; SEQ ID NOS: 1 and 3, respectively), VP16 (F10779 and F12668primers; SEQ ID NOS: 1 and 5, respectively), p53 (F12669 and F12505primers; SEQ ID NOS: 8 and 4, respectively), T antigen (F12670 andF12505 primers; SEQ ID NOS: 9 and 4, respectively), and SV40pA (F12016and F561 primers; SEQ ID NOS: 6 and 7, respectively) elements containingtopoisomerase sites at the appropriate ends. Topoisomerase was used tocreate the covalently linked, double stranded constructs GAL4+p53+SV40pAand VP16+T antigen+SV40pA, and the resultant ligation products were usedas templates for PCR amplification.

Purified GAL4+p53+SV40pA and VP16+T antigen+SV40pA PCR constructs wereco-transfected with a lacZ reporter gene (pGene/lacZ plasmid;Invitrogen) into CHO cells (6 well plate, 1×10⁵ cells/well). In parallelexperiments, the use of plasmid vectors containing the expressionconstructs was examined, as was the use of PCR reaction mixturescontaining the unpurified constructs. Control reactions were performedusing GAL4+pA and VP16+pA without inserts (negative controls) orp53+VP16 (positive control). Cells were lysed 48 hr after transfectionand reporter gene activity was measured using a β-galactosidase assaykit.

A high level of reporter gene activity was detected with the positivecontrol (FIG. 3, sample 3) and in the sample co-transfected with thereporter gene and the linear GAL4+p53+SV40pA and VP16+T antigen+SV40pAconstructs (FIG. 3, sample 4). Low level activity (but greater than thatof the negative controls; samples 5, 6, 8 and 9) was detected when theplasmid version of the constructs was used (FIG. 3, sample 1). Low levelactivity was also observed in the sample co-transfected with theunpurified, PCR-generated prey and bait constructs (sample 7). Theseresults demonstrate that a method of the invention can be used toprepare constructs useful for performing a two hybrid assay.

EXAMPLE 3 Generation, Purification, and Transfection of Gene-Specificd-siRNA and TOPO-Mediated Generation of Templates and Production ofDouble-Stranded RNA for Use in RNA Interference Analysis

Exemplary product literature is provided below that describes thegeneration, purification, and transfection of gene-specific d-siRNA foruse in RNA interference analysis, TOPO-mediated generation of templatesand production of double-stranded RNA for use in RNA interferenceanalysis. All catalog numbers provided below correspond to InvitrogenCorporation products, Carlsbad, Calif., unless otherwise noted. See alsoU.S. Ser. No. 10/902,704, entitled “Compositions and Methods forPreparing Short RNA Molecules and Other Nucleic Acids,” filed Jul. 30,2004, which is incorporated herein by reference.

D-siRNA Generation and Transfection Procedure

Produce dsRNA

Follow the guidelines to generate dsRNA. If you are using the BLOCK-iT™Complete Dicer RNAi Kit, refer to the BLOCK-iT™ RNAi TOPO® TranscriptionKit manual for instructions to generate dsRNA.

Perform the dicing reaction

1. Set up the following dicing reaction: 10X Dicer Buffer 30 μlRNase-Free Water up to 210 μl Purified dsRNA (60 μg) 1-150 μl BLOCK-iT ™Dicer Enzyme 60 μl (1 U/μl) Total volume 300 μl

-   -   2. Mix reaction gently and incubate for 14-18 hours at 37° C.    -   3. Add 6 μl of 50× Dicer Stop Solution.    -   4. Check integrity of the d-siRNA, if desired. Proceed to purify        d-siRNA.

Purify d-siRNA

-   1. To each 300 μl dicing reaction, add 300 μl of RNA Binding Buffer    containing 1% (v/v) β-mercaptoethanol followed by 300 μl of    isopropanol. Mix well by pipetting up and down 5 times.-   2. Apply half the sample (˜450 μl) to the RNA Spin Cartridge, and    centrifuge at 14,000×g for 15 seconds at room temperature. Save the    flow-through.-   3. Transfer the RNA Spin Cartridge to an siRNA Collection Tube and    repeat Step 2, using the other half of the dicing reaction sample    (˜450 μl). Save the flow-through.-   4. Transfer the flow-through from Step 2 to the siRNA Collection    Tube containing the flow-through from Step 3. Add 600 μl of    isopropanol and mix well by pipetting up and down 5 times.-   5. Apply one-third of the sample (˜500 μl) to a new RNA Spin    Cartridge. Centrifuge at 14,000×g for 15 seconds at room    temperature. Discard the flow-through.-   6. Repeat Step 5 twice, applying one-third of the remaining sample    (˜500 μl) to the RNA Spin Cartridge each time.-   7. Add 500 μl of 1×RNA Wash Buffer to the RNA Spin Cartridge, and    centrifuge at 14,000×g for 15 seconds at room temperature. Discard    the flow-through.-   8. Repeat Step 7.-   9. Centrifuge the RNA Spin Cartridge at 14,000×g for 1 minute at    room temperature.-   10. Remove the RNA Spin Cartridge from the Wash Tube and place it in    an RNA Recovery Tube.-   11. Add 30 μl of RNase-Free Water to the RNA Spin Cartridge. Let    stand at room temperature for 1 minute, then centrifuge the RNA Spin    Cartridge at 14,000×g for 2 minutes at room temperature to elute the    d-siRNA.-   12. Add 30 μl of RNase-Free Water to the RNA Spin Cartridge and    repeat Step 11, eluting the d-siRNA into the same RNA Recovery Tube.-   13. Add 1.2 μl of 50×RNA Annealing Buffer to the eluted d-siRNA.-   14. Quantitate the yield of d-siRNA by spectrophotometry. Aliquot    and store the d-siRNA at −80° C.

Transfect d-siRNA

Follow the procedure below to transfect cells using Lipofectamine™ 2000.Refer to later table for the appropriate reagent amounts and volumes toadd for different tissue culture formats.

-   -   1. One day before transfection, plate cells in growth medium        without antibiotics such that they will be 30-50% confluent at        the time of transfection.    -   2. For each transfection sample, prepare d-siRNA:Lipofectamine™        2000 complexes as follows:        -   a. Dilute d-siRNA in the appropriate amount of Opti-MEM® I            Reduced Serum Medium without serum. Mix gently.        -   b. Mix Lipofectamine™ 2000 gently before use, then dilute            the appropriate amount in Opti-MEM® 1. Mix gently and            incubate for 5 minutes at room temperature.        -   c. After the 5 minute incubation, combine the diluted            d-siRNA with the diluted Lipofectamine™ 2000. Mix gently and            incubate for 20 minutes at room temperature.    -   3. Add the d-siRNA:Lipofectamine™ 2000 complexes to each well        containing cells and medium. Mix gently by rocking the plate        back and forth.    -   4. Incubate the cells at 37° C. in a CO2 incubator until they        are ready to assay for gene knockdown.        Control Reaction

If you have purchased the BLOCK-iT™ Complete Dicer RNAi Kit, werecommend using the control template and control PCR primers includedwith the kit to produce dsRNA (see the BLOCK-iT™ RNAi TOPO®Transcription Kit manual for details). Once you have produced dsRNA, usethis dsRNA as a control in your dicing, purification, and transfectionexperiments.

Kit Contents and Storage

Types of Kits

The BLOCK-iT™ Complete Dicer RNAi Kit is also supplied with theBLOCK-iT™ RNAi TOPO® Transcription Kit and the BLOCK-iT™ RNAi TOPO®Transcription Kit manual. Product Catalog No. BLOCK-iT ™ Dicer RNAiTransfection K3600-01 Kit BLOCK-iT ™ Complete Dicer RNAi Kit K3650-01

Kit Components

The BLOCK-iT™ Dicer RNAi Kits include the following components. For adetailed description of the contents of each component, see laterdescription. For a detailed description of the contents of the BLOCK-iT™RNAi TOPO® Transcription Kit, see the BLOCK-iT™ RNAi TOPO® TranscriptionKit manual. Catalog no. Component K3600-01 K3650-01 BLOCK-iT ™ DicerEnzyme Kit ✓ ✓ BLOCK-iT ™ RNAi Purification Kit ✓ ✓ Lipofectamine ™ 2000Reagent ✓ ✓ BLOCK-iT ™ RNAi TOPO ® Transcription Kit ✓

Shipping/Storage

The BLOCK-iT™ Dicer RNAi Kits are shipped as described below. Uponreceipt, store each item as detailed below. For more detailedinformation about the reagents supplied with the BLOCK-iT™ RNAi TOPO®Transcription Kit, refer to the BLOCK-iT™ RNAi TOPO® Transcription Kitmanual. Box Component Shipping Storage 1 BLOCK-iT ™ Dicer Dry ice −20°C. Enzyme Kit 2 BLOCK-iT ™ RNAi Room temperature Room temperaturePurification Kit 3 Lipofectamine ™ 2000 Wet ice +4° C. (do not freeze)Reagent 4-6 BLOCK-iT ™ RNAi BLOCK-iT ™ BLOCK-iT ™ TOPO ® TOPO ®Transcription Kit TOPO ® Linker Kit Linker Kit and BLOCK- and BLOCK-iT ™iT ™ RNAi Transcription RNAi Transcription Kit: −20° C. Kit: Dry iceBLOCK-iT ™ RNAi BLOCK-iT ™ RNAi Purification Kit: Room Purification Kit:temperature Room temperatureBLOCK-iT ™ Dicer Enzyme Kit

The following reagents are included with the BLOCK-iT™ Dicer Enzyme Kit(Box 1). Store the reagents at −20° C. Reagent Composition AmountBLOCK-iT ™ Dicer Enzyme 1 U/μl in a buffer 300 μl 10X Dicer Buffer 150μl 50X Dicer Stop Buffer 0.5 mM EDTA, pH 8.0 30 μl RNase-Free Water —1.5 ml

One unit of BLOCK-iT™ Dicer enzyme cleaves 1 μg of double-stranded RNA(dsRNA) in 16 hours at 37° C.

BLOCK-iT™ RNAi Purification Kit

The following reagents are included with the BLOCK-iT™ RNAi PurificationKit (Box 2). Store reagents at room temperature. Use caution whenhandling the RNA Binding Buffer. Note: Catalog no. K3650-01 includes twoboxes of BLOCK-iT™ RNAi Purification reagents. One box is supplied withthe BLOCK-iT™ RNAi TOPO® Transcription Kit for purification of sense andantisense single-stranded RNA (ssRNA). The second box is supplied forpurification of diced siRNA (d-siRNA). Reagent Composition Amount RNABinding Buffer 1.8 ml 5X RNA Wash Buffer 2.5 ml RNase-Free Water — 800μl RNA Spin Cartridges — 10 RNA Recovery Tubes — 10 siRNA CollectionTubes* — 5 50X RNA Annealing Buffer 500 mM Tris-HCl, pH 8.0 50 μl 1 MNaCl  50 mM EDTA, pH 8.0*siRNA Collection Tubes are used for purification of d-siRNA only, andare not required for the purification of the ssRNA.

The RNA Binding Buffer supplied in the BLOCK-iT™ RNAi Purification Kitcontains guanidine isothiocyanate. This chemical is harmful if it comesin contact with the skin or is inhaled or swallowed. Always wear alaboratory coat, disposable gloves, and goggles when handling solutionscontaining this chemical.

Do not add bleach or acidic solutions directly to solutions containingguanidine isothiocyanate or sample preparation waste. Guanidineisothiocyanate forms reactive compounds and toxic gases when mixed withbleach or acids.

Lipofectamine™ 2000 Reagent

Each BLOCK-iT™ Dicer RNAi Kit includes Lipofectamine™ 2000 Reagent (Box3) for high efficiency transfection of d-siRNA into mammalian cells.Lipofectamine™ 2000 Reagent is supplied as follows:

-   Size: 0.75 ml-   Concentration: 1 mg/ml-   Storage: +4° C.; do not freeze

BLOCK-iT™ RNAi TOPO® Transcription Kit

The BLOCK-iT™ Complete Dicer RNAi Kit (Catalog no. K3650-01) includesthe BLOCK-iT™ RNAi TOPO® Transcription Kit to facilitate production ofdouble-stranded RNA (dsRNA) from your gene of interest. Refer to theBLOCK-iT™ RNAi TOPO® Transcription Kit manual for a detailed descriptionof the reagents provided with the kit and instructions to produce dsRNA.

Accessory Products

The products listed in this section may be used with the BLOCK-iT™ DicerRNAi Kits.

Accessory Products

Some of the reagents supplied in the BLOCK-iT™ Dicer RNAi Kits as wellas other products suitable for use with the kit are available separatelyfrom Invitrogen. Item Amount Catalog no. BLOCK-iT ™ RNAi TOPO ® 5 genesK3500-01 Transcription Kit Lipofectamine ™ 2000 Reagent 0.75 ml11668-027 1.5 ml 11668-019 Opti-MEM ® I Reduced Serum 100 ml 31985-062Medium 500 ml 31985-070 Phosphate-Buffered Saline 500 ml 10010-023(PBS), pH 7.4 4% E-Gel ® Starter Pak 9 gels and Base G5000-04 20%Novex ® TBE Gel 1 box EC63152BOX 10 bp DNA Ladder 50 μg 10821-015 β-GalAssay Kit 100 reactions K1455-01

Overview

The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ CompleteDicer RNAi Kit facilitate generation of purified diced siRNA duplexes(d-siRNA) that are suitable for use in RNAi analysis of a target gene inmammalian cells. Both kits contain the BLOCK-iT™ Dicer Enzyme for dicingdsRNA, reagents to purify the d-siRNA, and an optimized transfectionreagent for highly efficient delivery of d-siRNA to mammalian cells.

The BLOCK-iT™ Complete Dicer RNAi Kit also includes the BLOCK-iT™ RNAiTOPO® Transcription Kit to facilitate high-yield generation of purifieddsRNA. For more information, refer to the BLOCK-iT™ RNAi TOPO®Transcription Kit manual. This manual is supplied with the BLOCK-iT™Complete Dicer RNAi Kit.

Advantages of the BLOCK-iT™ Dicer RNAi Transfection Kit

Using the BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™Complete Dicer RNAi Kit to generate d-siRNA for RNAi analysis inmammalian provides the following advantages:

Provides a cost-effective means to enzymatically generate a pool ofd-siRNA that cover a larger portion of the target gene (e.g. 500 bp to 1kb) without the need for expensive chemical synthesis of siRNA.

Provides the BLOCK-iT™ Dicer Enzyme and an optimized protocol tofacilitate generation of the highest yields of d-siRNA from a dsRNAsubstrate.

Includes BLOCK-iT™ RNAi Purification reagents for efficient purificationof d-siRNA. Purified d-siRNA can be quantitated, enabling highlyreproducible RNAi analysis.

Includes the Lipofectamine™ 2000 Reagent for the highest efficiencytransfection in a wide variety of mammalian cell lines.

Purpose of this Manual

This manual provides the following information:

-   -   1. A description of the components in the BLOCK-iT™ Dicer RNAi        Transfection Kit and an overview of the pathway by which d-siRNA        facilitates gene knockdown in mammalian cells.    -   2. Guidelines to produce dsRNA corresponding to the target gene.        For detailed instructions to produce dsRNA, refer to the        BLOCK-iT™ RNAi TOPO® Transcription Kit manual.    -   3. Guidelines and instructions to use the BLOCK-iT™ Dicer Enzyme        to cleave dsRNA to generate a complex pool of d-siRNA.    -   4. Instructions to purify d-siRNA.    -   5. Guidelines and instructions to transfect purified d-siRNA        into mammalian cells using Lipofectamine™ 2000 Reagent for RNAi        studies.

The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ CompleteDicer RNAi Kit are designed to help generate d-siRNA for use in RNAianalysis in mammalian cell lines. Although the kits have been designedto help generate d-siRNA representing a particular target sequence inthe simplest, most direct fashion, use resulting d-siRNA for RNAianalysis assumes that users are familiar with the principles of genesilencing and transfection in mammalian systems. We highly recommendthat users possess a working knowledge of the RNAi pathway andlipid-mediated transfection.

For more information about the RNAi pathway in mammalian cells, refer topublished reviews (Elbashir, S. M., et al., Methods 26:199-213 (2002);McManus, M. T. and Sharp, P. A., Nature Rev. Genet. 3:737-747 (2002)).

BLOCK-iT™ Dicer RNAi Kit

Components of the BLOCK-iT™ Dicer RNAi Kit

The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ CompleteDicer RNAi Kit facilitate generation and delivery of purified d-siRNAduplexes into mammalian cells for RNAi analysis. The kits contain threemajor components:

-   -   1. The BLOCK-iT™ Dicer Enzyme and optimized reagents for        production of high yields of d-siRNA from a dsRNA substrate. For        more information about how the BLOCK-iT™ Dicer Enzyme works,        below.    -   2. The BLOCK-iT™ RNAi Purification reagents for silica-based        column purification of d-siRNA, and an RNA Annealing Buffer to        stabilize d-siRNA duplexes for long-term storage.    -   3. Lipofectamine™ 2000 Reagent for high-efficiency transfection        of d-siRNA into a wide range of mammalian cell types and cell        lines for RNAi analysis.

If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, note that thekit also includes a control expression plasmid containing the lacZ geneand PCR primers that may be used to generate control lacZ dsRNA. Thecontrol lacZ dsRNA may be used in a dicing and purification reaction togenerate purified lacZ d-siRNA. Co-transfecting the purified lacZd-siRNA and the control expression plasmid into mammalian cells providea means to assess the RNAi response in your cell line by assaying forknockdown of β-galactosidase. In addition, the lacZ d-siRNA can be usedas a negative control for non-specific off-target effects in your RNAistudies.

If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, note that thekit includes 2 boxes of BLOCK-iT™ RNAi Purification reagents. One box isintended for purification of dsRNA, while the second box is intended forpurification of d-siRNA. The protocols to purify dsRNA and d-siRNAdiffer significantly from one another. When purifying d-siRNA, be sureto use the purification procedure provided in this manual. To purifydsRNA, use the purification procedure provided in the BLOCK-iT™ RNAiTOPO® Transcription Kit manual.

Generating d-siRNA Using the Kit

Using the reagents supplied in the kit, you will perform the followingsteps to generate pure d-siRNA that is ready for transfection into themammalian cell line of interest.

-   1. Use dsRNA representing your target sequence (generated with the    BLOCK-iT™ RNAi TOPO® Transcription Kit) in a reaction with the    BLOCK-iT™ Dicer enzyme to generate d-siRNA.-   2. Purify the d-siRNA using the purification reagents supplied in    the kit. Quantitate the yield of purified d-siRNA obtained.-   3. Transfect d-siRNA into the mammalian cell line of interest using    Lipofectamine™ 2000 Reagent.

The RNAi Pathway and How Dicer Works

The RNAi Pathway

RNAi describes the phenomenon by which dsRNA induces potent and specificinhibition of eukaryotic gene expression via the degradation ofcomplementary messenger RNA (mRNA), and is functionally similar to theprocesses of post-transcriptional gene silencing (PTGS) or cosuppressionin plants (Cogoni, C., et al., Antonie Van Leeuwenhoek 65:205-209(1994); Napoli, C., et al., Plant Cell 2:279-289 (1990); Smith, C. J.,et al., Mol. Gen. Genet. 224:477-481 (1990); van der Krol, A. R., etal., Plant Cell 2:291-299 (1990)) and quelling in fungi (Cogoni, C. andMacino, G., Nature 399:166-169 (1999); Cogoni, C. and Macino, G., Proc.Natl. Acad. Sci. USA 94:10233-10238 (1997); Romano, N. and Macino, G.,Mol. Microbiol. 6:3343-3353 (1992)). In plants, the PTGS response isthought to occur as a natural defense against viral infection ortransposon insertion (Anandalakshmi, R., et al., Proc. Natl. Acad. Sci.USA 95:13079-13084 (1998); Jones, A. L., et al., EMBO J. 17:6385-6393(1998); Li, W. X. and Ding, S. W., Curr. Opin. Biotechnol. 12:150-154(2001); Voinnet, O., et al., Proc. Natl. Acad. Sci. USA 96:14147-14152(1999)).

In eukaryotic organisms, dsRNA produced in vivo or introduced bypathogens is processed into 21-23 nucleotide double-stranded shortinterfering RNA duplexes (siRNA) by an enzyme called Dicer (Bernstein,E., et al., Nature 409:363-366 (2001); Ketting, R. F., et al., GenesDev. 15:2654-2659 (2001)). The siRNA then incorporate into theRNA-induced silencing complex (RISC), a second enzyme complex thatserves to target cellular transcripts complementary to the siRNA forspecific cleavage and degradation (Hammond, S. M., et al., Nature404:293-296 (2000); Nykanen, A., et al., Cell 107:309-321 (2001)).

For more information about the RNAi pathway and the mechanism of genesilencing, refer to recent reviews (Bosher, J. M. and Labouesse, M.,Nature Cell Biol. 2:E31-E36 (2000); Hannon, G. J., Nature 418:244-251(2002); Plasterk, R. H. A. and Ketting, R. F., Genet. Dev. 10:562-567(2000); Zamore, P. D., Biol. 8:746-750 (2001)).

Performing RNAi Analysis in Mammalian Cells

A number of kits including the BLOCK-iT™ RNAi TOPO® Transcription Kitnow exist to facilitate in vitro production of dsRNA that is targeted toa particular gene of interest. The dsRNA may be introduced directly intosome invertebrate organisms or cell lines, where it functions to triggerthe endogenous RNAi pathway resulting in inhibition of the target gene.Long dsRNA duplexes cannot be used directly for RNAi analysis in mostsomatic mammalian cell lines because introduction of long dsRNA intothese cell lines induces a non-specific, interferon-mediated response,resulting in shutdown of translation and initiation of cellularapoptosis (Kaufman, R. J., Proc. Natl. Acad. Sci. USA 96:11693-11695(1999)). To avoid triggering the interferon-mediated host cell response,dsRNA duplexes of less than 30 nucleotides must be introduced into cells(Stark, G. R., et al., Annu. Rev. Biochem. 67:227-264 (1998)). Foroptimal results in gene knockdown studies, the size of the dsRNAduplexes (i.e. siRNA) introduced into mammalian cells is further limitedto 21-23 nucleotides.

Using the Kit for RNAi Analysis

The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ CompleteDicer RNAi Kit facilitate in vitro production of a complex pool of 21-23nucleotide siRNA duplexes that is targeted to a particular gene ofinterest. The kits use a recombinant human Dicer enzyme (see below formore information) to cleave a long dsRNA substrate (produced with theBLOCK-iT™ RNAi TOPO® Transcription Kit) into a pool of 21-23 nucleotided-siRNA that may be transfected into mammalian cells. Introduction ofd-siRNA into the cells then triggers the endogenous RNAi pathway,resulting in inhibition of the target gene. For a diagram of theprocess, see FIG. 10.

BLOCK-iT™ Dicer Enzyme

BLOCK-iT™ Dicer is a recombinant human enzyme (Myers, J. W., et al.,Nat. Biotechnol. 21:324-328 (2003); Provost, P., et al., EMBO J.21:5864-5874 (2002)) that cleaves long dsRNA processively into 21-23nucleotide d-siRNA duplexes with 2 nucleotide 3′ overhangs. The Dicerenzyme is a member of the RNase III family of double-strandedRNA-specific endonucleases, and consists of an ATP-dependent RNAhelicase domain, a Piwi/Argonaute/Zwille (PAZ) domain, two RNase IIIdomains, and a dsRNA-binding domain (Bernstein, E., et al., Nature409:363-366 (2001); Zamore, P. D., Biol. 8:746-750 (2001)). In additionto its role in the generation of siRNA, Dicer is also involved in theprocessing of short temporal RNA (stRNA) (Hutvagner, G., et al., Science293:811-813 (2001); Ketting, R. F., et al., Genes Dev. 15:2654-2659(2001)) and microRNA (mRNA) (Carrington, J. C. and Ambros, V., Science301:336-338 (2003)) from stable hairpin or stem-loop precursors.

Experimental Outline

The table below outlines the desired steps when using the BLOCK-iT™Dicer RNAi Kits to generate, purify, and transfect your d-siRNA ofinterest. Step Action 1 Produce dsRNA from your target gene. 2 Use thedsRNA in a reaction with the BLOCK-iT ™ Dicer enzyme to generate d-siRNA. 3 Purify d-siRNA using the BLOCK-iT ™ RNAi Purification Reagents.4 Transfect purified d-siRNA into your mammalian cell line of interestusing Lipofectamine ™ 2000 Reagent. 5 Assay for inhibition of targetgene expression using your method of choice.

Methods

Generating Double-Stranded RNA (dsRNA)

Introduction

Before you can use the BLOCK-iT™ Dicer Enzyme to produce shortinterfering RNA (siRNA), you should generate double-stranded RNA (dsRNA)substrate representing your target sequence of interest. Guidelines andrecommendations to generate dsRNA are provided below.

For optimal, high-yield production of dsRNA, we recommend using theBLOCK-iT™ RNAi TOPO® Transcription Kit available from Invitrogen(Catalog no. K3500-01). The BLOCK-iT™ RNAi TOPO® Transcription Kitsupplies the reagents necessary to generate T7 promoter-based DNAtemplates from any Taq-amplified PCR product, then use these templatesin in vitro transcription reactions to generate sense and antisense RNAtranscripts. The kit also includes reagents to enable purification andannealing of the RNA transcripts to produce high yields of dsRNA thatare ready-to-use in the dicing reaction.

For detailed protocols and guidelines to generate dsRNA from your targetgene sequence, refer to the BLOCK-iT™ RNAi TOPO® Transcription Kitmanual. This manual is supplied with the BLOCK-iT™ Complete Dicer RNAiKit.

Choosing the Target Sequence

When performing RNAi analysis, your choice of target sequence cansignificantly affect the degree of gene knockdown observed. In addition,the size of the target sequence and the resulting dsRNA can affect theyields of d-siRNA produced. Consider the following factors when choosingyour target sequence.

Select a target sequence that covers a reasonable portion of the gene ofinterest and that does not contain regions of strong homology with othergenes.

Limit the size of the target sequence. Although smaller or larger targetsequences are possible, we recommend limiting the initial targetsequence to a size range of 500 bp to 1 kb for the following reasons.

-   -   a) This balances the risk of including regions of strong        homology between the target gene and other genes that could        result in non-specific off-target effects during RNAi analysis        with the benefits of using a more complex pool of siRNA.    -   b) When producing sense and antisense transcripts of the target        template, the highest transcription efficiencies are obtained        with transcripts in the 500 bp to 1 kb size range. Target        templates outside this size range transcribe less efficiently,        resulting in lower yields of dsRNA.    -   c) Double-stranded RNA that is under 1 kb in size is efficiently        diced. Larger dsRNA substrates can be used but yields may        decline as the size increases.

The BLOCK-iT™ Dicer RNAi Kits have been used successfully to knock downgene activity with dsRNA substrates ranging from 150 bp to 1.3 kb insize.

Factors to Consider When Generating dsRNA

If you are using your own method or another kit to produce dsRNA,consider the following factors when generating your dsRNA. These factorswill influence the yields of d-siRNA produced from the dicing reaction.

Amount of dsRNA desired for dicing: We use 60 μg of dsRNA in a typical300 μl dicing reaction to recover 12-18 μg of d-siRNA afterpurification. This amount of d-siRNA is generally sufficient totransfect approximately 150 wells of cells plated in a 24-well format.You should have an idea of the scale and scope of your RNAi experimentto determine how much dsRNA you will need to dice.

If you wish to dice less than 60 μg of dsRNA, you will need to scaledown the dicing reaction proportionally.

Concentration of dsRNA: The amount of dsRNA in a dicing reaction shouldnot exceed half the reaction volume; therefore, the concentration ofyour dsRNA should be ≧400 ng/μl if you wish to dice 60 μg of dsRNA.

Buffering of dsRNA: We recommend storing your dsRNA sample in a bufferedsolution containing 1 mM EDTA and no more than 100 mM salt (i.e. TEBuffer at pH 7-8 or 1×RNA Annealing Buffer). This helps to stabilize thedsRNA and provides the optimal environment for efficient cleavage by theDicer Enzyme.

If you have used the BLOCK-iT™ RNAi TOPO® Transcription Kit to producedsRNA, your dsRNA sample will be in 1X RNA Annealing Buffer (10 mMTris-HCl, 20 mM NaCl, 1 mM EDTA, pH 8.0).

The quality of your dsRNA: To obtain the highest yields of d-siRNA, werecommend using purified dsRNA in the dicing reaction.

Once you have generated your purified dsRNA, we recommend saving analiquot of the dsRNA for future gel analysis. We generally use agaroseor polyacrylamide gel electrophoresis to assess the success of thedicing reaction by comparing an aliquot of the dicing reaction to analiquot of the dsRNA substrate.

Performing the Dicing Reaction

Once you have produced your target dsRNA, you will perform an in vitrodicing reaction using the reagents supplied in the BLOCK-iT™ DicerEnzyme Kit (Box 1) to generate d-siRNA duplexes of 21-23 nucleotides insize.

BLOCK-iT™ Dicer Enzyme Activity

One unit of BLOCK-iT™ Dicer Enzyme cleaves 1 μg of dsRNA in 16 hours at37° C. Note that the Dicer enzyme does not cleave dsRNA to d-siRNA with100% efficiency, i.e. dicing 1 μg of dsRNA does not generate 1 μg ofd-siRNA. Under these optimal reaction conditions, the Dicer enzymecleaves dsRNA to d-siRNA with an efficiency of approximately 25-35%. Forexample, dicing 60 μg of dsRNA in a 300 μl dicing reaction typicallyyields 12-18 μg of d-siRNA following purification.

For best results, we recommend following the dicing procedure exactly asdescribed as the reaction conditions have been optimized to provide thehighest mass yield of d-siRNA under the most efficient dicingconditions.

It is possible to use more than 60 μg of dsRNA in a 300 μl dicingreaction; however, the BLOCK-iT™ Dicer Enzyme becomes less efficientunder these conditions. Although you may generate a higher mass yield ofd-siRNA, the % yield of d-siRNA will decrease.

Do not increase the amount of BLOCK-iT™ Dicer Enzyme used in the dicingreaction (to greater than 60 units in a 300 μl reaction) or increase thelength of the dicing reaction (to greater than 18 hours). Under eitherof these conditions, the BLOCK-iT™ Dicer Enzyme can bind to d-siRNA andcleave the 21-23 nt duplexes into smaller products, resulting in loweryields of d-siRNA.

Amount of dsRNA to Use

For a typical 300 μl dicing reaction, you will need 60 μg of targetdsRNA. If you want to dice less than 60 μg of dsRNA, scale down theentire reaction proportionally.

The total volume of dsRNA added should not exceed half the volume of thereaction. Thus, for best results, make sure that the startingconcentration of your dsRNA is ≧400 ng/[μl.

Positive Control

If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, and haveperformed all of the recommended control reactions using the controlreagents supplied in the BLOCK-iT™ RNAi TOPO® Transcription portion ofthe kit, you should have purified dsRNA representing a 1 kb portion ofthe lacZ gene. We recommend setting up a separate dicing andpurification reaction using the control lacZ dsRNA. You can thenco-transfect the resulting purified lacZ d-siRNA and the pcDNA™1.2V5-GW/lacZ control plasmid supplied with the kit into your mammaliancell line as a positive control for the RNAi response in that cell line.Alternatively, you may use the lacZ d-siRNA as a negative control fornon-specific, off-target effects in your cell line.

When performing the dicing reaction and subsequent purification ofd-siRNA, take precautions to avoid RNase contamination.

Use RNase-free sterile pipette tips and supplies for all manipulations.

Use DEPC-treated solutions as necessary.

Wear gloves when handling reagents and solutions, and when performingreactions.

Materials Needed

Have the following reagents on hand before beginning:

-   -   1. Purified dsRNA (>400 ng/μl in 1×RNA Annealing Buffer or TE        Buffer, pH 7-8)    -   2. BLOCK-iT™ Dicer Enzyme (1 U/μl; supplied with the kit, Box 1;        keep at −20° C. until immediately before use)    -   3. 10× Dicer Buffer (supplied with the kit, Box 1)    -   4. RNase-Free Water (supplied with the kit, Box 1)    -   5. 50× Dicer Stop Buffer (supplied with the kit, Box 1)

Dicing Procedure

Follow the procedure below to perform the dicing reaction. Make surethat the volume of dsRNA added does not exceed half the volume of thereaction (i.e. ≦150 μl).

1. Set up a 300 μl dicing reaction on ice using the following reagentsin the order shown. Reagent Sample 10X Dicer Buffer 30 μl RNase-FreeWater up to 210 μl Purified dsRNA (60 μg) 1-150 μl BLOCK-iT ™ DicerEnzyme (1 U/μl) 60 μl Total volume 300 μl

-   2. Mix reaction gently and incubate for 14-18 hours at 37° C.-   Do not incubate the reaction for longer than 18 hours as this may    result in a lower yield of d-siRNA due to cleavage of d-siRNA by the    Dicer enzyme.-   3. Add 6 μl of 50× Dicer Stop Solution to the reaction.-   4. Check the integrity of your d-siRNA, if desired.-   5. Proceed to purify the d-siRNA (see Purifying Diced siRNA    (d-siRNA),) or store the dicing reaction overnight at −20° C.

Checking the Integrity of d-siRNA

You may verify the integrity of your d-siRNA using polyacrylamide oragarose gel electrophoresis, if desired. We suggest running an aliquotof your dicing reaction (0.5-1 μl of a 300 μl reaction; equivalent to100-200 ng of dsRNA) on the appropriate gel and comparing it to analiquot of your starting dsRNA. Be sure to include an appropriatemolecular weight standard. We generally use the following gels andmolecular weight standard:

Agarose gel: 4% E-Gel® ((Invitrogen, Catalog no. G5000-04)

Polyacrylamide gel: 20% Novex® TBE Gel (Invitrogen, Catalog no.EC63152BOX)

Molecular weight standard: 10 bp DNA Ladder (Invitrogen, Catalog no.10821-015)

When analyzing an aliquot of the dicing reaction by gel electrophoresis,we generally see the following:

A predominant band of approximately 21-23 nt representing the d-siRNA.

4% E-Gel®: A high molecular weight smear representing uncleaved dsRNAand partially cleaved products. Generally, this band does not resolvewell on an agarose gel and runs close to the well.

Novex® 20% TBE Gel: A high molecular weight band and a smearrepresenting uncleaved dsRNA and partially cleaved products. The dsRNAband generally resolves better on a polyacrylamide gel.

If the band representing d-siRNA is weak or if you do not see a band,see Troubleshooting for tips to troubleshoot your dicing reaction.

Example of Expected Results

In this experiment, purified dsRNA representing a 1 kb region of thelacZ gene was generated following the recommended protocols and usingthe reagents supplied in the BLOCK-iT™ RNAi TOPO® Transcription Kit. ThelacZ dsRNA was diced using the procedure outlined below. Aliquots of thedicing reaction (equivalent to 200 ng of dsRNA) and the initial dsRNAsubstrate were analyzed on a 4% E-Gel®.

Results are shown in FIG. 11: A prominent band representing d-siRNA ofthe expected size is clearly visible in the dicing reaction sample (lane3). This band is not visible in the initial dsRNA substrate sample (lane2). Lane 1. 10 bp DNA Ladder. Lane 2. 200 ng purified lacZ dsRNA. Lane3. 200 ng lacZ dicing reaction.

Purifying Diced siRNA (d-siRNA)

Introduction

This section provides guidelines and instructions to purify the d-siRNAproduced in the dicing reaction. Use the BLOCK-iT™ RNAi Purificationreagents (Box 2) supplied with the kit.

Before proceeding to transfection, note that you should purify thed-siRNA produced in the dicing reaction to remove contaminating longdsRNA duplexes. Transfection of unpurified d-siRNA can trigger theinterferon-mediated response and cause host cell shutdown and cellularapoptosis. When purifying d-siRNA, follow the purification procedureprovided below exactly as instructed. This procedure is optimized toallow removal of contaminating long dsRNA and recovery of high yields ofd-siRNA.

Experimental Outline

To purify d-siRNA, you will:

-   1. Add RNA Binding Buffer and isopropanol to the dicing reaction to    denature the proteins and to enable the contaminating dsRNA to bind    to the column.-   2. Add half the volume of the sample to an RNA spin cartridge. The    dsRNA binds to the silica-based membrane in the cartridge, and the    d-siRNA and denatured proteins flow through the cartridge. Save the    flow-through.-   3. Transfer the RNA spin cartridge to an siRNA Collection Tube and    add the remaining sample to the RNA spin cartridge. Repeat Step 2.    Save the flow-through.-   4. Pool the flow-throughs from Step 2 and Step 3 in the siRNA    Collection Tube and add isopropanol to the sample to enable the    d-siRNA to bind to the column.-   5. Add the sample to a second RNA spin cartridge. The d-siRNA bind    to the membrane in the cartridge.-   6. Wash the membrane-bound d-siRNA to eliminate residual RNA Binding    Buffer, isopropanol, and any remaining impurities.-   7. Elute the d-siRNA from the RNA spin cartridge with water.-   8. Add 50×RNA Annealing Buffer to the eluted d-siRNA to stabilize    the d-siRNA for storage.

For an illustration of the d-siRNA purification process, see FIG. 12.

Advance Preparation

Before using the BLOCK-iT™ RNA Purification reagents for the first time,add 10 ml of 100% ethanol to the entire amount of 5×RNA Wash Buffer toobtain a 1X RNA Wash Buffer (total volume=12.5 ml). Place a check in thebox on the 5×RNA Wash Buffer label to indicate that the ethanol wasadded. Store the 1×RNA Wash Buffer at room temperature.

The RNA Binding Buffer contains guanidine isothiocyanate. This chemicalis harmful if it comes in contact with the skin or is inhaled orswallowed. Always wear a laboratory coat, disposable gloves, and goggleswhen handling solutions containing this chemical.

Do not add bleach or acidic solutions directly to solutions containingguanidine isothiocyanate or sample preparation waste. Guanidineisothiocyanate forms reactive compounds and toxic gases when mixed withbleach or acids.

Materials Needed

Have the following materials on hand before beginning:

-   -   1.Dicing reaction (from Step 5)    -   2. RNA Binding Buffer (supplied with the kit, Box 2)    -   3. β-mercaptoethanol    -   4. Isopropanol    -   5. RNA Spin Cartridges (supplied with the kit, Box 2; two for        each sample)    -   6. siRNA Collection Tube (supplied with the kit, Box 2)    -   7. 1×RNA Wash Buffer (see Advance Preparation, above)    -   8. RNase-Free Water (supplied with the kit, Box 2)    -   9. RNA Recovery Tube (supplied with the kit, Box 2)    -   10. 50×RNA Annealing Buffer (supplied with the kit, Box 2)    -   11. RNase-free supplies

d-siRNA Purification Procedure

Use this procedure to purify d-siRNA produced from dicing 60 μg of dsRNAin a 300 μl reaction volume (see Step 5). If you have digested <60 μg ofdsRNA and have scaled down the volume of your dicing reaction, scaledown the volume of your purification reagents proportionally. Forexample, if you have digested 30 μg of dsRNA in a 150 μl dicingreaction, scale down the volume of purification reagents used by half.

Before beginning, remove the amount of RNA Binding Buffer needed and addβ-mercaptoethanol to a final concentration of 1% (v/v). Use fresh anddiscard any unused solution.

-   1. To each dicing reaction (˜300 μl volume), add 300 μl of RNA    Binding Buffer containing 1% (v/v) β-mercaptoethanol followed by 300    μl of isopropanol to obtain a final volume of 900 μl. Mix well by    pipetting up and down 5 times.-   2. Apply half of the sample (˜450 μl) to the RNA Spin Cartridge.    Centrifuge at 14,000×g for 15 seconds at room temperature.-   3. Transfer the RNA spin cartridge to an siRNA Collection Tube. Save    the flow-through containing d-siRNA from Step 2.-   4. Apply the remaining half of the sample (˜450 μl) to the RNA Spin    Cartridge. Centrifuge at 14,000×g for 2 minutes at room temperature.-   5. Remove the RNA Spin Cartridge from the siRNA Collection Tube and    discard. Save the flow-through containing d-siRNA.-   6. Transfer the flow-through from Step 2 (˜450 μl) to the siRNA    Collection Tube containing the flow-through from Step 4 (˜450 μl) to    obtain a final volume of ˜900 μl. Add 600 μl of isopropanol to the    sample to obtain a final volume of 1.5 ml. Mix well by pipetting up    and down.-   7. Apply one-third of the sample (˜500 μl) to a new RNA Spin    Cartridge. Centrifuge at 14,000×g for 15 seconds at room    temperature. Discard the flow-through.-   8. Repeat Step 7 twice, applying one-third of the remaining sample    (˜500 μl) to the RNA Spin Cartridge each time.-   9. Add 500 μl of 1×RNA Wash Buffer to the RNA Spin Cartridge    containing bound d-siRNA. Centrifuge at 14,000×g for 15 seconds at    room temperature. Discard the flow-through.-   10. Repeat the wash step (Step 9).-   11. Centrifuge the RNA Spin Cartridge at 14,000×g for 1 minute at    room temperature to remove residual 1×RNA Wash Buffer from the    cartridge and to dry the membrane.-   12. Remove the RNA Spin Cartridge from the Wash Tube, and place it    in an RNA Recovery Tube.-   13. Add 30 μl of RNase-Free Water to the RNA Spin Cartridge. Let    stand at room temperature for 1 minute, then centrifuge the RNA Spin    Cartridge at 14,000×g for 2 minutes at room temperature to elute the    d-siRNA. Proceed to Step 14.-   14. Add 30 μl of RNase-Free Water to the RNA Spin Cartridge and    repeat Step 13, eluting the d-siRNA into the same RNA Recovery Tube.    The total volume of eluted d-siRNA is 60 μl.-   15. Add 1.2 μl of the 50×RNA Annealing Buffer to the eluted d-siRNA    to obtain a final concentration of 1×RNA Annealing Buffer. Adding    RNA Annealing Buffer to the sample increases the stability of the    d-siRNA.-   16. Proceed to quantitate the concentration of your purified d-siRNA    (see Determining the Purity and Concentration of d-siRNA, below).-   17. Store the purified d-siRNA at −80° C. Depending on the amount of    d-siRNA produced and your downstream application, you may want to    aliquot the d-siRNA before storage at −80° C.

When using the d-siRNA, avoid repeated freezing and thawing as d-siRNAcan degrade with each freeze/thaw cycle.

Determining the Purity and Concentration of d-siRNA

Use the procedure below to determine the purity and concentration ofyour purified d-siRNA.

-   1. Dilute an aliquot of the purified d-siRNA 20-fold into 1×RNA    Annealing Buffer in a total volume appropriate for your quartz    cuvettes and spectrophotometer.-   2. Measure OD at A260 and A280 in a spectrophotometer. Blank the    sample against 1×RNA Annealing Buffer.-   3. Calculate the concentration of the d-siRNA by using the following    equation: d-siRNA concentration (μg/ml)=A260×Dilution factor (20)×40    μg/ml.-   4. Calculate the yield of the d-siRNA by using the following    equation: d-siRNA yield (μg)=d-siRNA concentration (μg/ml)×vol. of    d-siRNA (ml)-   5. Evaluate the purity of the purified d-siRNA by determining the    A260/A280 ratio. For optimal purity, the A260/A280 ratio should    range from 1.9-2.2.

Verifying the Quality of Your d-siRNA

You may verify the quality of your purified d-siRNA using polyacrylamideor agarose gel electrophoresis, if desired. We suggest running a smallaliquot of your purified-siRNA (0.5-1 μl) on the appropriate gel andcomparing it to an aliquot of your dicing reaction (equivalent to100-200 ng of dsRNA). Be sure to include an appropriate molecular weightstandard. For recommended gels and a molecular weight standard, wegenerally use the same gels and molecular weight standard that we use toanalyze the quality of the dicing reaction.

If the band representing purified d-siRNA is weak or if you do not see aband, see Troubleshooting for tips to purify your d-siRNA.

Example of Expected Results

In this experiment, the lacZ d-siRNA generated in the dicing reactiondepicted above were purified using the procedure described above.Aliquots of the purified lacZ d-siRNA (80 ng) and the lacZ dicingreaction (equivalent to 200 ng of dsRNA) were analyzed on a 4% E-Gel@.

Results are demonstrated in FIG. 13: A prominent band representingpurified-siRNA of the expected size is clearly visible in lane 3. Nocontaminating dsRNA or other high molecular weight products remain inthe purified d-siRNA sample. Lane 1. 10 bp DNA Ladder, Lane 2. 200 nglacZ dicing reaction, Lane 3. 80 ng purified lacZ d-siRNA.

The typical yield of d-siRNA obtained from dicing 60 μg of dsRNA (500 bpto 1 kb in size) in a 300 μl dicing reaction ranges from 12-18 μg, witha concentration of 200-300 ng/μl. Note that yields may vary depending onthe size and quality of the dsRNA

Transfecting Cells

Introduction

Once you have purified your d-siRNA, you may perform RNAi analysis bytransfecting the d-siRNA into the mammalian cell line of interest, andassaying for inhibition of expression from your target gene. Thissection provides general guidelines and protocols to transfect yourpurified d-siRNA into mammalian cells using the Lipofectamine™ 2000Reagent (Box 3) supplied with the kit. Suggested transfection conditionsare provided as a starting point. You will need to optimize transfectionconditions to obtain the best results for your target gene and mammaliancell line.

You must transfect mammalian cells with purified d-siRNA. Note thattransfecting cells with unpurified d-siRNA containing contaminating longdsRNA (i.e. with material directly taken from the dicing reaction) cantrigger the interferon-mediated cellular response, resulting in hostcell shutdown and cellular apoptosis.

Factors Affecting Gene Knockdown Levels

A number of factors can influence the degree to which expression of yourgene of interest is reduced (i.e. gene knockdown) in an RNAi experimentincluding:

-   -   1. Transfection efficiency    -   2. Transcription rate of the target gene of interest    -   3. Stability of the target protein    -   4. Growth characteristics of your mammalian cell line

Take these factors into account when designing your transfection andRNAi experiments.

Lipofectamine™ 2000 Reagent

The Lipofectamine™ 2000 Reagent supplied with the kit is a cationiclipid-based formulation suitable for the transfection of nucleic acidsincluding d-siRNA and siRNA into eukaryotic cells (Ciccarone, V., etal., Focus 21:54-55 (1999); Gitlin, L., et al., Nature 418:430-434(2002); Yu, J. Y., et al, Proc. Nat. Acad. Sci. USA 99:6047-6052(2002)). Using Lipofectamine™ 2000 to transfect d-siRNA into eukaryoticcells offers the following advantages:

-   -   1. Provides the highest transfection efficiency in many cell        types    -   2. Is the most widely used transfection reagent for delivery of        d-siRNA or siRNA into eukaryotic cells (Gitlin, L., et al.,        Nature 418:430-434 (2002); Yu, J. Y., et al., Proc. Nat. Acad.        Sci. USA 99:6047-6052 (2002))    -   3. d-siRNA-Lipofectamine™ 2000 complexes can be added directly        to cells in culture medium in the presence of serum.    -   4. Removal of complexes, medium change, or medium addition        following transfection are not required, although complexes can        be removed after 4-6 hours without loss of activity.

Lipofectamine™ 2000 is also available separately from Invitrogen.

Important Guidelines

Follow these guidelines when transfecting siRNA into mammalian cellsusing Lipofectamine™ 2000:

-   1. Cell density: For optimal results, we recommend plating cells    such that they will be 30-50% confluent at the time of transfection.    Gene knockdown levels are generally assayed 24-72 hours following    transfection. Transfecting cells at a lower density allows a longer    interval between transfection and assay time, and minimizes the loss    of cell viability due to cell overgrowth. Depending on the nature of    the target gene, higher or lower cell densities may be suitable with    optimization of conditions.-   2. For optimal results, use Opti-MEM® I Reduced Serum Medium    (Invitrogen, Catalog no. 31985-062) to dilute Lipofectamine™ 2000    and d-siRNA prior to complex formation.-   3. Do not include antibiotics in media used during transfection as    this will reduce transfection efficiency and cause cell death.

Materials to Have on Hand

Have the following materials on hand before beginning:

-   -   1. Mammalian cell line of interest (make sure that cells are        healthy and greater than 90% viable before transfection)    -   2. Purified d-siRNA of interest (>40 ng/μl)    -   3. If you have diced 60 μg of dsRNA, the typical yield of        d-siRNA obtained after purification is 12-18 μg at a        concentration of 200-300 ng/μl)    -   4. Positive control, if desired (see below)    -   5. Lipofectamine™ 2000 Reagent (supplied with the kit; store at        +4° C. until use)    -   6. Opti-MEM® I Reduced Serum Medium (Invitrogen, Catalog no.        31985-062; pre-warmed)    -   7. Sterile tissue culture plates and other tissue culture        supplies

Positive Control

If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, and have dicedthe control lacZ dsRNA, two options exist to use the resulting purifiedlacZ d-siRNA for RNAi analysis:

-   1. Use the lacZ d-siRNA as a negative control for non-specific    off-target effects.-   2. Use the lacZ d-siRNA as a positive control to assess the RNAi    response in your cell line by co-transfecting the lacZ d-siRNA and    the pcDNA™ 1.2/V5-GW/lacZ reporter plasmid supplied with the kit    into your mammalian cells using Lipofectamine™ 2000. Assay for    knockdown of β-galactosidase expression 24 hours post-transfection    using Western blot analysis or activity assay.

Transfection conditions (i.e. cell density and reagent amounts) varyslightly when d-siRNA and plasmid DNA are co-transfected into mammaliancells. For details, see Co-transfecting d-siRNA and Plasmid DNA.

Transfection Procedure

Use this procedure to transfect mammalian cells using Lipofectamine™2000. Refer to the table in Recommended Reagent Amounts and Volumes,below for the appropriate reagent amounts and volumes to add fordifferent tissue culture formats. Use the recommended Lipofectamine™2000 amounts as a starting point for your experiments, and optimizeconditions for your cell line and d-siRNA.

-   1. One day before transfection, plate cells in the appropriate    amount of growth medium without antibiotics such that they will be    30-50% confluent at the time of transfection.-   2. For each transfection sample, prepare d-siRNA:Lipofectamine™ 2000    complexes as follows:    -   (a) Dilute d-siRNA in the appropriate amount of Opti-MEMO® I        Reduced Serum Medium without serum. Mix gently.    -   (b) Mix Lipofectamine™ 2000 gently before use, then dilute the        appropriate amount in Opti-MEM® I Reduced Serum Medium. Mix        gently and incubate for 5 minutes at room temperature. Combine        the diluted Lipofectamine™ 2000 with the diluted d-siRNA within        30 minutes. Longer incubation times may decrease activity.    -   (c) After the 5 minute incubation, combine the diluted d-siRNA        with the diluted Lipofectamine™ 2000. Mix gently and incubate        for 20 minutes at room temperature to allow the        d-siRNA:Lipofectamine™ 2000 complexes to form. The solution may        appear cloudy, but this will not inhibit transfection.-   3. Add the d-siRNA:Lipofectamine™ 2000 complexes to each well    containing cells and medium. Mix gently by rocking the plate back    and forth.-   4. Incubate the cells at 37° C. in a CO₂ incubator for 24-96 hours    as appropriate until they are ready to assay for gene knockdown. It    is not necessary to remove the complexes or change the medium;    however, growth medium may be replaced after 4-6 hours without loss    of transfection activity.

Recommended Reagent Amounts and Volumes

The table below lists the recommended reagent amounts and volumes to useto transfect cells in various tissue culture formats. Use therecommended amounts of d-siRNA (see column 4) and Lipofectamine™ 2000(see column 6) as a starting point for your experiments, and optimizeconditions for your cell line and target gene. With automated,high-throughput systems, larger complexing volumes are recommended fortransfections in 96-well plates. Lipofectamine ™ Relative d-siRNA 2000(μl) Lipofectamine ™ Surface Volume (μg) and d-siRNA and 2000 Area ofDilution Amounts (μl) Dilution Amounts (μl) Culture (vs. 24- PlatingVolume for Volume for Vessel well) Medium (μl) Optimization (μl)Optimization 96-well 0.2 100 μl 20 ng in  5-50 ng 0.6 μl in 0.2-1.0 μl25 μl 25 μl 24-well 1 500 μl 50 ng in 20-200 ng 1 μl in 50 μl 0.5-1.5 μl50 μl  6-well 5  2 ml 250 ng in 100 ng-1 μg 5 μl in   2.5-6 μl 250 μl250 μl

Optimizing Transfection

To obtain the highest transfection efficiency and low non-specificeffects, optimize transfection conditions by varying the cell density(from 30-50% confluence) and the amounts of d-siRNA (see column 5) andLipofectamine™ 2000 (see column 7) as suggested in the table above. Forcell lines that are particularly sensitive to transfection-mediatedcytotoxicity (e.g. HeLa, HTT1080), use the lower amounts ofLipofectamine™ 2000 suggested in the table above.

What You Should See

When performing RNAi experiments using d-siRNA, we generally observeinhibition of the gene of interest within 24 to 96 hours aftertransfection. The degree of gene knockdown depends on the time of assay,stability of the protein of interest, and on the other factors. Notethat 100% gene knockdown is generally not observed, but >95% is possiblewith optimized conditions.

Co-Transfecting d-siRNA and Plasmid DNA

If you are using the lacZ d-siRNA as a positive control to assess theRNAi response in your cell line, you will co-transfect the lacZ d-siRNAand the pcDNA™ 1.2/V5-GW/lacZ reporter plasmid into the mammalian cellline and assay for inhibition of β-galactosidase expression after 24hours. When co-transfecting d-siRNA and plasmid DNA, follow theprocedure on the previous page with the following exceptions:

Plate cells such that they will be 90% confluent at the time oftransfection.

Refer to the table below for the recommended amount of d-siRNA (seecolumn 3) and plasmid DNA (see column 4) to transfect in a particulartissue culture format.

We generally transfect twice the mass of plasmid DNA as d-siRNA.

Use the recommended Lipofectamine™ 2000 amounts in the table below (seecolumn 6) as a starting point, and optimize conditions for your cellline if desired. To optimize conditions, vary the amount ofLipofectamine™ 2000 as suggested in the table below (see column 7).Lipofectamine ™ Lipofectamine ™ Volume Nucleic 2000 2000 of Plasmid Acid(μl) and Amounts (μl) Culture Plating d-siRNA DNA Dilution Dilution forVessel Medium (μg) (μg) Volume Volume (μl) Optimization 96-well 100 μl20 ng  40 ng 25 μl 0.6 μl in 25 μl 0.2-1.0 μl 24-well 500 μl 50 ng 100ng 50 μl   2 μl in 50 μl 0.5-2.0 μl  6-well  2 ml 250 ng 500 ng 250 μl  10 μl in 250 μl  2.5-10 μl

Assaying for β-Galactosidase Expression

If you perform RNAi analysis using the control lacZ d-siRNA, you mayassay for β-galactosidase expression and knockdown by Western blotanalysis or activity assay using cell-free lysates (Miller, J. H.,Experiments in Molecular Genetics (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory (1972)). Invitrogen offers the β-gal Antiserum(Catalog no. R901-25) and the β-Gal Assay Kit (Catalog no. K1455-01) forfast and easy detection of β-galactosidase expression.

The β-galactosidase protein expressed from the pcDNA™ 1.2/V5-GW/lacZcontrol plasmid is fused to a V5 epitope and is approximately 119 kDa insize. If you are performing Western blot analysis, you may also use theAnti V5 Antibodies available from Invitrogen (e.g. Anti-V5-HRP Antibody;Catalog no. R961-25 or Anti-V5-AP Antibody, Catalog no. R962-25) fordetection.

Examples of Expected Results

Introduction

This section provides some examples of results obtained from RNAiexperiments performed with d-siRNA generated using the BLOCK-iT™Complete Dicer RNAi Kit. The first example depicts knockdown ofexpression of a reporter gene, and the second example depicts knockdownof expression of the endogenous lamin A/C gene.

Example of Expected Results: Knockdown of a Reporter Gene

In this experiment, d-siRNA targeting two reporter genes (i.e.luciferase and lacZ) and an endogenous gene (i.e. lamin A/C) wasgenerated following the recommended protocols and using the reagentssupplied in the BLOCK-iT™ Complete Dicer RNAi Kit.

GripTite™ 293 MSR cells (Invitrogen, Catalog no. R795-07) were grown to90% confluence. Individual wells in a 24-well plate were transfectedusing Lipofectamine™ 2000 Reagent with 100 ng each of lacZ andluciferase-containing reporter plasmids. In some wells, the reporterplasmids were co-transfected with 50 ng of purified lacZ, luciferase, orlamin A/C d-siRNA. Cell lysates were prepared 24 hours aftertransfection and assayed for luciferase and β-galactosidase activity.Activities were normalized to those of the reporter plasmids alone.

Results are shown in FIG. 14: Potent and specific inhibition is evidentfrom luciferase and lacZ-derived d-siRNA. Note that in this experiment,lamin A/C d-siRNA serves as a negative control and does not inhibitluciferase or B3-galactosidase expression.

Introduction of d-siRNA into mammalian cells can, in some cases lead toa slight induction of gene expression, as is observed with13-galactosidase and luciferase expression upon transfection of lamind-siRNA.

Example of Expected Results: Knockdown of an Endogenous Gene

In this experiment, dsRNA representing a 1 kb region of the lamin A/Cgene and the luciferase gene were produced following the recommendedprotocols and using reagents supplied in the BLOCK-iT™ RNAi TOPO®Transcription Kit. The target sequences chosen for the lamin A/C andluciferase genes were as described by (Elbashir, S. M., et al., Nature411:494-498 (2001)). The resulting dsRNA were used as substrates togenerate lamin A/C and luciferase d-siRNA following the recommendedprotocols and using the reagents supplied in the BLOCK-iT™ CompleteDicer RNAi Kit. 104751 50 ng each of lamin A/C and luciferase d-siRNA aswell as 4 pmoles each (about 50 ng) of synthetic lamin A/C andluciferase siRNA (21 nucleotide duplexes) were transfected into A549(human lung carcinoma) cells plated in a 24-well plate usingLipofectamine™ 2000. Cell lysates were prepared 48 hourspost-transfection and analyzed by Western blot using an Anti-Lamin A/CAntibody (1:1000 dilution, BD Biosciences, Catalog no. 612162) and anAnti-β-Actin Antibody (1:5000 dilution, Abcam, Catalog no. ab6276).

Results are shown in FIG. 15: Only the lamin A/C-specific d-siRNA (lane2) and siRNA (lane 4) were able to inhibit expression of the lamin A/Cgene, while no lamin A/C gene knockdown was observed with the luciferased-siRNA (lane 3) or siRNA (lane 5). In addition, the degree of lamin A/Cgene blocking achieved using the lamin A/C d-siRNA was similar to thatachieved with the well-characterized, chemically-synthesized siRNA.Lane 1. Mock transfection, Lane 2. 50 ng lamin A/C d-siRNA, Lane 3. 50ng luciferase d-siRNA, Lane 4. 4 pmol lamin A/C siRNA, Lane 5. 4 pmolluciferase siRNA.

Troubleshooting

Use the information in this section to troubleshoot your dicing,purification, and transfection experiments.

Dicing Reaction

The table below lists some potential problems and possible solutionsthat may help you troubleshoot the dicing reaction. Problem ReasonSolution Weak band Poor quality Generate dsRNA using the representing d-dsRNA BLOCK-iT ™ RNAi TOPO ® siRNA Transcription Kit (refer to theobserved on BLOCK-iT ™ RNAi TOPO ® a poly- Transcription Kit manual foracrylamide or instructions). agarose gel Verify the concentration of(i.e. low yield your dsRNA. of d-siRNA) Didn't use Use 60 μg of dsRNA ina enough dsRNA 300 μl dicing reaction. If you are in the dicing dicingless dsRNA, scale down the reaction entire dicing reactionproportionally. Make sure that the amount of dsRNA added does not exceedhalf the reaction volume (i.e. concentration of initial dsRNAsubstrate >400 ng/μl). dsRNA was Make sure that the dsRNA degradedsample is in a buffer containing 1 mM EDTA (i.e. TE Buffer, pH 7-8 or 1XRNA Annealing Buffer). Avoid repeated freeze/thaw cycles. Aliquot thedsRNA and store at −80° C. Incubated the Do not incubate the dicingreaction for dicing reaction longer than 18 hours. for longer than 18hours Incubated the Incubate the dicing reaction at 37° C. dicingreaction for 14-18 hours. for less than 14 hours Smear with Used toomuch Follow the recommended procedure to molecular BLOCK-iT ™ set up thedicing reaction. Do not use weight <21 nt Dicer Enzyme more than 60units of BLOCK-iT ™ observed on a in the dicing Dicer Enzyme in a 300 μlreaction. poly- reaction acrylamide gel Incubated the Do not incubatethe dicing reaction for dicing reaction longer than 18 hours. for longerthan 18 hours Sample Use RNase-free supplies contaminated and solutions.with RNase Wear gloves when handling reagents and setting up the dicingreaction. No d-siRNA dsRNA was Make sure that the dsRNA produceddegraded sample is in a buffer containing 1 mM EDTA (i.e. TE Buffer, pH7-8 or 1X RNA Annealing Buffer). Avoid repeated freeze/thaw cycles.Aliquot the dsRNA and store at −80° C. Sample was Use RNase-freesupplies contaminated and solutions. with RNase Wear gloves whenhandling reagents and setting ssRNA used as If you have used to theBLOCK-iT ™ substrate RNAi TOPO ® Transcription Kit to generate sense andantisense ssRNA, you should anneal the ssRNA to generate dsRNA prior todicing.

Purifying d-siRNA

The table below lists some potential problems and possible solutionsthat may help you troubleshoot the purification procedure. ProblemReason Solution Low yield of Eluted d-siRNA Elute d-siRNA from the RNASpin purified d- from the RNA Spin Cartridge using water. siRNACartridge using TE obtained Buffer Concentration of d- siRNA incorrectlydetermined Sample Dilute sample in 1X diluted into water RNA AnnealingBuffer for for spectrophotometry. spectrophotometry Sample Blank sampleagainst 1X blanked against RNA Annealing Buffer. water No d-siRNA Forgotto add Add 10 ml of ethanol to the 5X obtained ethanol to the 5X RNAWash Buffer (2.5 ml) to RNA Wash Buffer obtain a 1X RNA Wash Buffer.Forgot to add You should add isopropanol to the isopropanol to thecombined flow-throughs from the combined flow- first RNA Spin Cartridgeto enable throughs from the the d-siRNA to bind to the second first RNASpin RNA Spin Cartridge. Cartridge Forgot to keep flow- Keep theflow-throughs from the throughs from the first RNA Spin Cartridge (Steps3 first RNA Spin and 5). The flow-throughs contain Cartridge thed-siRNA. dsRNA Forgot to add You should add RNA Binding present inisopropanol to the Buffer containing 1% (v/v) β- purified d- dicingreaction mercaptoethanol and isopropanol to siRNA the dicing reaction todenature the sample proteins and enable the dsRNA to bind the first RNASpin Cartridge. Added the mixture You should add the mixture containingthe flow- containing the flow-through and through and isopropanol fromthe first RNA Spin isopropanol from Cartridge (Step 6) to a second RNAthe first RNA Spin Spin Cartridge as the first RNA Cartridge (Step 6)Spin Cartridge contains bound back onto the first dsRNA. RNA SpinCartridge A260/A280 Sample was not Wash the RNA Spin Cartridge ratio notin washed with 1X containing bound d-siRNA twice the 1.9-2.2 RNA WashBuffer with 1X RNA Wash Buffer (see range Steps 9 and 10). RNA SpinCartridge Centrifuge RNA Spin Cartridge at containing bound d- 14,000 ×g for 1 minute at room siRNA not temperature to remove residual 1Xcentrifuged to RNA Wash Buffer and to dry the remove residual 1Xmembrane (see Step 11). RNA Wash Buffer

Transfection and RNAi Analysis

The table below lists some potential problems and possible solutionsthat may help you troubleshoot your transfection and knockdownexperiment. Problem Reason Solution Low levels Low transfection of geneefficiency knockdown Antibiotics Do not add antibiotics observed addedto the media to the media during transfection. during transfection Cellswere Plate cells such that confluent at the time they will be 30-50%confluent at of transfection the time of transfection. Not enoughIncrease the amount of d-siRNA transfected d-siRNA transfected. Notenough Optimize the Lipofectamine ™ transfection conditions for your2000 used cell line by varying the amount of Lipofectamine ™ 2000 used.Didn't wait long Repeat the enough after transfection and wait for alonger transfection before period of time after transfection assayingfor gene before assaying for gene knockdown knockdown. Perform a timecourse of expression to determine the point at which the highest degreeof gene knockdown occurs. d-siRNA was degraded Make sure that the d-siRNA is stored in 1X RNA Annealing Buffer. Aliquot purified d- siRNAand avoid repeated freeze/thaw cycles. Cytotoxic Too much Optimize thetransfection effects Lipofectamine ™ conditions for your cell line byobserved 2000 Reagent varying the amount of after used Lipofectamine ™2000 Reagent transfection used. Cells transfected with Purify d-siRNAusing the RNAi unpurified d-siRNA Purification reagents supplied withthe kit. Transfecting unpurified d-siRNA is not recommended as thecontaminating dsRNA will cause host cell shutdown and apoptosis. No gened-siRNA was degraded knockdown d-siRNA Make sure that the d- observedwas stored in water siRNA is stored in 1X RNA Annealing Buffer. d-siRNAAliquot purified d- was repeatedly frozen siRNA and avoid repeated andthawed freeze/thaw cycles. Target region contains Select a larger targetregion or a no active siRNA different region. Non-specific Targetsequence Select a new target sequence. off-target contains strong Limitthe size range of the target gene homology to other sequence to 1 kb.knockdown genes observed

Product Qualification

Introduction The components of the BLOCK-iT™ Dicer RNAi Kits arequalified as described below.

Functional Qualification

The BLOCK-iT™ Dicer enzyme and RNAi Purification reagents arefunctionally qualified as follows:

-   1. The BLOCK-iT™ Dicer enzyme is diluted to 1 U/μl and tested (in    triplicate) in a dicing reaction following the procedure above using    lacZ dsRNA produced using the BLOCK-iT™ RNAi TOPO® Transcription    Kit. Each dicing reaction is assessed by analyzing an aliquot of the    reaction on a 20% Novex® TBE gel (Catalog no. EC63152BOX). The 10 bp    DNA Ladder (Catalog no. 10821-015) is included as a molecular weight    standard. Polyacrylamide gel analysis should demonstrate a minimal    amount of dsRNA remaining in the reaction and minimal to no    degradation of siRNA apparent.-   2. The dicing reactions are purified using the RNAi purification    reagents supplied in the kit and following the procedure above.    Purified d-siRNA is quantitated using spectrophotometry. The amount    of d-siRNA recovered should be at least 25%.

Lipofectamine™ 2000 Reagent

Lipofectamine™ 2000 is tested for the absence of microbial contaminationusing blood agar plates, Sabaraud dextrose agar plates, and fluidthioglycolate medium, and functionally by transfection of CHO-K1 cellswith a luciferase reporter-containing plasmid.

BLOCK-iT™ RNAi TOPO® Transcription Kit

Introduction

This quick reference sheet is provided for experienced users of thedsRNA generation procedure. If you are performing the TOPO® Linking,secondary amplification, transcription, purification, or annealing stepsfor the first time, follow the detailed protocols provided in themanual. We recommend using the pcDNA™ 1.2/V5-GW/lacZ plasmid and thecontrol PCR primers (lacZ Forward 2 and lacZ Reverse 2 primers) includedwith the kit to generate dsRNA. Step Action Produce the PCR 1. Amplifyyour sequence of interest using Platinum ® Taq product DNA polymeraseand your own protocol. End the PCR reaction with a final 7 minuteextension step. 2. Use agarose gel electrophoresis to check theintegrity and yield of your PCR product. Perform the 1. Set up thefollowing TOPO ® Linking reaction. TOPO ® Linking Your PCR product (≧20ng/μl) 1 μl reaction Salt Solution 1 μl Sterile water 3 μl BLOCK-iT ™T7-TOPO ® Linker 1 μl Total volume 6 μl 2. Mix reaction gently andincubate for 15 minutes at 37° C. 3. Place the reaction on ice andproceed directly to perform secondary amplification, below. Perform 1.Set up 2 PCR reactions - in each reaction, amplify 1 μl of secondary theTOPO ® Linking reaction using Platinum ® Taq DNA amplificationpolymerase and your own protocol. End the PCR reaction reactions to witha final 7 minute extension step. For PCR primers, use generate sense thefollowing: and antisense Sense template: use the BLOCK-iT ™ T7 Primerand DNA templates your gene-specific reverse primer Antisense template:use the BLOCK-iT ™ T7 Primer and your gene-specific forward primer 2.Use agarose gel electrophoresis to check the integrity and yield of yourPCR products. 3. Proceed to perform the RNA transcription reactions,next page. Perform the RNA 1. Set up two separate transcriptionreactions using either transcription the sense or antisense linear DNAtemplate. reaction to RNase-free water up to 21 μl generate sense 75 mMNTPs 8 μl and antisense DNA template (250 ng-1 μg) 1-10 μl ssRNA 100XTranscription buffer 4 μl BLOCK-iT ™ T7 Enzyme Mix 6 μl Total volume 40μl 2. Incubate the reaction at 37° C. for 2 hours. 3. Add 2 μl of DNaseI to each reaction. Incubate at 37° C. for 15 minutes. Purify the senseand 1. To each RNA transcription reaction, add 160 μl of RNA antisensetranscripts Binding Buffer containing 1% (v/v) β-mercaptoethanolfollowed by 100 μl of 100% ethanol. Mix well by pipetting up and down 5times. 2. Apply the sample to the RNA Spin Cartridge, and centrifuge at14,000 × g for 15 seconds at room temperature. Discard the flow-through.3. Add 500 μl of 1X RNA Wash Buffer to the RNA Spin Cartridge, andcentrifuge at 14,000 × g for 15 seconds at room temperature. Discard theflow-through. 4. Repeat Step 3. 5. Centrifuge the RNA Spin Cartridge at14,000 × g for 1 minute at room temperature. 6. Remove the RNA SpinCartridge from the Wash Tube, and place it in an RNA Recovery Tube. Add40 μl of RNase- free water to the RNA Spin Cartridge. Let stand at roomtemperature for 1 minute, then centrifuge the RNA Spin Cartridge at14,000 × g for 2 minutes at room temperature to elute the ssRNA. 7. Add40 μl of RNase-Free Water to the RNA Spin Cartridge and repeat Step 7,eluting the ssRNA into the same RNA Recovery Tube. Add 1.4 μl of 50X RNAAnnealing Buffer to the eluted ssRNA. 8. Quantitate the yield of ssRNAby spectrophotometry. Anneal the sense and 1. In a microcentrifuge tube,mix equal amounts of purified antisense transcripts sense and antisensessRNA. to produce 2. Heat 250 ml of water to boiling in a 500 ml glassbeaker, dsRNA remove from the heat, and set the beaker on the laboratorybench. 3. Place the tube containing the ssRNA mixture (in a tube float)in the glass beaker and allow the water to cool to room temperature for1-1.5 hours. 4. Aliquot and store the dsRNA at −80° C.

Kit Contents and Storage

Types of Kits

This manual is supplied with the products listed below.

The BLOCK-iT™ Complete Dicer RNAi Kit is also supplied with theBLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ Dicer RNAi Kitsmanual. Product Catalog no. BLOCK-iT ™ RNAi TOPO ® Transcription KitK3500-01 BLOCK-iT ™ Complete Dicer RNAi Kit K3650-01

Kit Components

The BLOCK-iT™ RNAi Kits include the following components. For adescription of the contents of the BLOCK-iT™ RNAi TOPO® Transcription

The BLOCK-iT™ Complete Dicer RNAi Kit also includes the BLOCK-iT™ DicerRNAi Transfection Kit. For a detailed description of the reagentssupplied in the BLOCK-iT™ Dicer RNAi Transfection Kit, refer to theBLOCK-iT™ Dicer RNAi Kits Catalog no. Component K3500-01 K3650-01BLOCK-iT ™ RNAi TOPO ® Transcription ✓ ✓ Kit BLOCK-iT ™ Dicer RNAiTransfection Kit ✓

Shipping/Storage

The BLOCK-iT™ RNAi TOPO® Transcription Kit is shipped as describedbelow. Upon receipt, store each item as detailed below. Box ComponentShipping Storage 1 BLOCK-iT ™ TOPO ® Dry ice −20° C. Linker Kit 2BLOCK-iT ™ RNAi Dry ice −20° C. Transcription Kit 3 BLOCK-iT ™ RNAi Roomtemperature Room Purification Kit temperature

BLOCK-iT™ TOPO® Linker Kit Reagents

The following reagents are supplied with the BLOCK-iT™ TOPO® Linker Kit(Box 1). Note that the user must supply Taq polymerase. Store thereagents at −20° C. Reagent Composition Amount BLOCK-iT ™ 0.1-1 ng/μldouble-stranded DNA in: 5 μl T7-TOPO ®X 50 mM Tris-HCl, pH 7.3 Linker100 mM NaCl 0.2 mM EDTA 0.9 mM DTT 45 μg/ml BSA 0.05% (v/v) Triton X-10040% (v/v) glycerol 10X PCR Buffer 100 mM Tris-HCl, pH 8.3 (at 42° C.) 75μl 500 mM KCl 25 mM MgCl₂ 0.01% gelatin 40 mM dNTPs 10 mM dATP 15 μl 10mM dTTP 10 mM dGTP 10 mM dCTP neutralized at pH 8.0 in water SaltSolution 1.2 M NaCl 10 μl 0.06 M MgCl₂ Sterile Water — 750 μl BLOCK-iT ™T7 75 ng/μl in TE Buffer, pH 8.0 10 μl Primer LacZ Forward 2 65 ng/μl inTE Buffer, pH 8.0 10 μl Primer LacZ Reverse 2 Primer 65 ng/μl in TEBuffer, pH 8.0 10 μl pcDNA ™ 1.2/ Lyophilized in TE Buffer, pH 8.0 10 μgV5-GW/lacZ control plasmid

Primer Sequences

The table below provides the sequence and the amount supplied of theprimers included in the kit. Primer Sequence Amount BLOCK-iT™ T75′-GATGACTCGTAATACGACTCACTA-3′ (SEQ ID NO. 48) 103 pmoles LacZ Forward 25′-ACCAGAAGCGGTGCCGGAAA-3′ (SEQ ID NO. 49) 105 pmoles LacZ Reverse 25′-CCACAGCGGATGGTTCGGAT-3′ (SEQ ID NO. 50) 106 pmoles

BLOCK-iT™ RNAi Transcription Kit Reagents

The following reagents are included with the BLOCK-iT™ RNAiTranscription Kit. Store reagents at −20° C. Reagent Composition AmountBLOCK-iT ™ T7 Enzyme  60 μl Mix 10X Transcription Buffer  40 μl 75 mMNTPs 18.75 mM ATP  80 μl 18.75 mM UTP 18.75 mM CTP 18.75 mM GTPneutralized at pH 8.0 in water RNase-Free Water — 800 μl DNase I 1 U/μlin  20 μl 20 mM sodium acetate, pH 6.5 5 mM CaCl₂ 0.1 mM PMSF 50% (v/v)glycerol

BLOCK-iT™ RNAi Purification Kit

The following reagents are included with the BLOCK-iT™ RNAi PurificationKit. Store reagents at room temperature. Use caution when handling theRNA Binding Buffer.

Catalog no. K3650-01 includes two boxes of BLOCK-iT™ RNAi Purificationreagents. One box is supplied with the BLOCK-iT™ RNAi TOPO®Transcription Kit for purification of the single-stranded RNA (ssRNA).The second box is supplied with the BLOCK-iT™ Dicer RNAi TransfectionKit for purification of diced siRNA (d-siRNA). Reagent CompositionAmount RNA Binding Buffer 1.8 ml 5X RNA Wash Buffer 2.5 ml RNase-FreeWater — 800 μl RNA Spin Cartridges — 10 RNA Recovery Tubes — 10 siRNACollection Tubes* — 5 50X RNA Annealing 500 mM Tris-HCl, pH 8.0 50 μlBuffer 1 M NaCl 50 mM EDTA, pH 8.0siRNA Collection Tubes are not required for the purification of thessRNA, and are used for purification of d-siRNA only.

The RNA Binding Buffer supplied in the BLOCK-iT™ RNAi Purification Kitcontains guanidine isothiocyanate. This chemical is harmful if it comesin contact with the skin or is inhaled or swallowed. Always wear alaboratory coat, disposable gloves, and goggles when handling solutionscontaining this chemical.

Do not add bleach or acidic solutions directly to solutions containingguanidine isothiocyanate or sample preparation waste. Guanidineisothiocyanate forms reactive compounds and toxic gases when mixed withbleach or acids.

Accessory Products

The table below provides ordering information for products availablefrom Invitrogen that are suitable for use with the BLOCK-iT™ RNAi TOPO®Transcription Kit. Item Amount Catalog no. BLOCK-iT ™ Dicer RNAi 5 genes× 150 K3600-01 Transfection Kit transfections each* Taq DNA Polymerase,Native 100 units 18038-018 500 units 18038-042 Taq DNA Polymerase,Recombinant 100 units 10342-053 500 units 10342-020 Platinum ® Taq DNAPolymerase 100 reactions 10966-018 250 reactions 10966-026 500 reactions10966-034 6% Novex ® TBE Gel 1 box EC6265BOX 0.16-1.77 kb RNA Ladder 75μg 15623-010*Based on transfection in 24-well plates.

Introduction

The BLOCK-iT™ RNAi TOPO® Transcription Kit facilitates rapid generationof T7 promoter-based DNA templates. Using the DNA templates and reagentssupplied with the kit, RNA transcripts are produced, purified, andannealed to generate double-stranded RNA (dsRNA). The resulting dsRNAmay be used directly for RNA interference (RNAi) analysis ininvertebrate systems and other systems lacking the interferon responseor as a substrate to produce short interfering RNA (siRNA) for RNAianalysis in mammalian cells.

Advantages of the BLOCK-iT™ RNAi TOPO® Transcription Kit

Use of the BLOCK-iT™ RNAi TOPO® Transcription Kit to facilitateproduction of dsRNA provides the following advantages:

-   -   1. The BLOCK-iT™ T7-TOPO® Linker provides a method to quickly        and easily add a T7 promoter to any existing Taq-amplified PCR        product without the need for new primers or subcloning.    -   2. Use of the TOPO® Linking Technology and secondary        amplification enables simultaneous production of linear DNA        templates that may be used directly for in vitro transcription        to generate sense and antisense transcripts. Creation of a T7        expression plasmid, bacterial transformation, and plasmid        purification are not required.    -   3. Separate transcription reactions using sense and antisense        templates allow precise quantitation of ssRNA concentration        prior to annealing.    -   4. Provides optimized purification reagents to obtain highly        pure sense and antisense transcripts that can be annealed to        generate an optimal yield of dsRNA. Double-stranded RNA can be        used directly for RNAi analysis in invertebrate systems or as a        substrate for the Dicer enzyme to generate siRNA.

This manual provides instructions and guidelines to:

-   1. Amplify your sequence of interest and use TOPO® Linking to join    the primary PCR product to the BLOCK-iT™ T7-TOPO® Linker.-   2. Use the appropriate primers to amplify the TOPO® Linked PCR    product to generate linear sense and antisense DNA templates.-   3. Use the linear sense and antisense DNA templates in transcription    reactions to generate sense and antisense single-stranded RNA    (ssRNA) transcripts of the sequence of interest.-   4. Purify the sense and antisense ssRNA transcripts and anneal them    to generate dsRNA. The resulting dsRNA may then be used in the    application of choice (e.g. RNAi analysis in invertebrate organisms    or as a substrate for “dicing” to produce d-siRNA for RNAi analysis    in mammalian cells).

For details and instructions to generate d-siRNA using Dicer, refer tothe BLOCK-iT™ Dicer RNAi Kits manual. This manual is supplied with theBLOCK-iT™ Dicer RNAi Transfection and Complete Dicer RNAi Kits.

The BLOCK-iT™ RNAi TOPO® Transcription Kit is designed to help yougenerate dsRNA for direct use in RNAi analysis in invertebrate systemsor as a substrate in a dicing reaction to produce d-siRNA for RNAianalysis in mammalian cells. Although the kit has been designed to helpyou generate dsRNA representing a particular target sequence in thesimplest, most direct fashion, use of the resulting dsRNA for RNAianalysis assumes that users are familiar with the mechanism of genesilencing and the techniques that exist to introduce dsRNA into theorganism or cell type of choice. We highly recommend that users possessa working knowledge of the RNAi pathway and the methodologies requiredto perform RNAi analysis in the organism or cell type of choice.

For more information about these topics, refer to published reviews(Bosher and Labouesse, 2000; Hannon, 2002; Plasterk and Ketting, 2000;Zamore, 2001). A variety of BLOCK-iT™ RNAi products are available fromInvitrogen to facilitate your RNAi analysis.

Description of the System

The BLOCK-iT™ RNAi TOPO® Transcription Kit facilitates generation of T7promoter-based DNA templates for in vitro transcription and productionof dsRNA, and consists of three major components:

-   1. The BLOCK-iT™ T7-TOPO® Linker for quick and easy creation of T7    promoter-based DNA templates for in vitro transcription. Using TOPO®    Linking Technology, the BLOCK-iT™ T7-TOPO® Linker may be linked to    any Taq-amplified PCR product. The linked PCR product is then    amplified to generate a linear DNA template.-   2. BLOCK-iT™ RNAi Transcription reagents for generation of sense and    antisense ssRNA transcripts from your T7-based, linear DNA template.    The reagents include an optimized T7 Enzyme Mix for highly efficient    production of ssRNA.-   3. The BLOCK-iT™ RNAi Purification reagents for silica-based column    purification of sense and antisense ssRNA transcripts, and an RNA    Annealing Buffer to stabilize dsRNA duplexes for long-term storage.

The BLOCK-iT™ RNAi TOPO® Transcription Kit also includes a controlexpression plasmid containing the lacZ gene and PCR primers that may beused as controls to generate dsRNA. Once generated, the lacZ dsRNA maybe used for the following types of RNAi analysis:

Invertebrate Systems

As a negative control for non-specific gene knockdown in anyinvertebrate system. The lacZ dsRNA is not suitable for use as apositive control to knock down β-galactosidase expression from thecontrol pcDNA™ 1.2/V5-GW/lacZ plasmid in any invertebrate system. Thisis because expression of the lacZ gene from the control plasmid iscontrolled by the human cytomegalovirus (CMV) promoter, and thispromoter is not active in most invertebrate systems.

Mammalian Systems

As a negative control for non-specific gene knockdown or as a positivecontrol for knockdown of β-galactosidase expression from the pcDNA™1.2/V5-GW/lacZ reporter plasmid. Note that to perform RNAi analysis inmammalian cells, the lacZ dsRNA should first be “diced” to generated-siRNA. For details, refer to the BLOCK-iT™ Dicer RNAi Kits manual.

Generating dsRNA Using the BLOCK-iT™ RNAi TOPO® Transcription Kit

You will perform the following steps to generate dsRNA using theBLOCK-iT™ RNAi TOPO® Transcription Kit. For a diagram, see FIG. 16illustrating the major steps necessary to generate dsRNA using theBLOCK-iT™ RNAi TOPO® Transcription System.

-   1. Amplify your sequence of interest using Taq polymerase.-   2. Perform a TOPO® Linking reaction to link your PCR product to the    BLOCK-iT™ T7-TOPO® Linker containing the T7 promoter.-   3. Using a combination of the BLOCK-iT™ T7 Primer (supplied with the    kit) and your gene-specific forward or reverse primer, amplify the    TOPO® Linked PCR product with Taq polymerase to produce linear sense    and antisense DNA templates.-   4. Use the sense and antisense DNA templates and the reagents    supplied in the kit in an in vitro transcription reaction to produce    sense and antisense RNA transcripts, respectively.-   5. Purify the sense and antisense RNA transcripts using the RNAi    Purification reagents supplied in the kit.-   6. Quantitate the yield of purified sense and antisense ssRNA    transcripts, and anneal equal amounts of each single-stranded    transcript to form dsRNA.

How TOPO® Linking Works

How Topoisomerase I Works

Topoisomerase I from Vaccinia virus binds to duplex DNA at specificsites and cleaves the phosphodiester backbone after 5′-CCCTT in onestrand (Shuman, 1991). The energy from the broken phosphodiesterbackbone is conserved by formation of a covalent bond between the 3′phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) oftopoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme cansubsequently be attacked by the 5′ hydroxyl of the original cleavedstrand, reversing the reaction and releasing topoisomerase (Shuman,1994). TOPO® Linking exploits this reaction to efficiently join PCRproducts to the BLOCK-iT™ T7-TOPO® Linker.

TOPO® Linking

The BLOCK-iT™ T7-TOPO® Linker is supplied linearized with:

-   -   A single 3 thymidine (T) overhang for TA Cloning®    -   Topoisomerase I covalently bound to the linker (this is referred        to as “activated linker”)

Taq polymerase has a nontemplate-dependent terminal transferase activitythat adds a single deoxyadenosine (A) to the 3′ ends of PCR products.The linear BLOCK-iT™ T7-TOPO® linker supplied in this kit has a single,overhanging 3′ deoxythymidine (T) residue. This allows PCR products toligate efficiently with the linker.

TOPO® Linking as shown in FIG. 17 exploits the ligation activity oftopoisomerase I by providing an “activated” linearized TA linker(Shuman, 1994). Ligation of the linker with a PCR product containing 3′A-overhangs is very efficient and occurs spontaneously with maximumefficiency at 37° C. within 15 minutes.

The RNAi Pathway

RNAi describes the phenomenon by which dsRNA induces potent and specificinhibition of eukaryotic gene expression via the degradation ofcomplementary messenger RNA (mRNA), and is functionally similar to theprocesses of post-transcriptional gene silencing (PTGS) or cosuppressionin plants (Cogoni et al., 1994; Napoli et al., 1990; Smith et al., 1990;van der Krol et al., 1990) and quelling in fungi (Cogoni and Macino,1999; Cogoni and Macino, 1997; Romano and Macino, 1992). In plants, thePTGS response is thought to occur as a natural defense against viralinfection or transposon insertion (Anandalakshmi et al., 1998; Jones etal., 1998; Li and Ding, 2001; Voinnet et al., 1999).

In eukaryotic organisms, dsRNA produced in vivo or introduced bypathogens is processed into 21-23 nucleotide double-stranded shortinterfering RNA duplexes siRNA)by an enzyme called Dicer, a member ofthe RNase III family of double-stranded RNA-specific endonucleases(Bernstein et al., 2001; Ketting et al., 2001). The siRNA thenincorporate into the RNA-induced silencing complex (RISC), a secondenzyme complex that serves to target cellular transcripts complementaryto the siRNA for specific cleavage and degradation (Hammond et al.,2000; Nykanen et al., 2001).

For more information about the RNAi pathway and the mechanism of genesilencing, refer to reviews (Bosher and Labouesse, 2000; Hannon, 2002;Plasterk and Ketting, 2000; Zamore, 2001).

Using the Kit for RNAi Analysis

The BLOCK-iT™ RNAi TOPO® Transcription Kit facilitates in vitroproduction of dsRNA that is targeted to a particular gene of interest.The long dsRNA is introduced into the appropriate organism or cells,where the endogenous Dicer enzyme processes the dsRNA into siRNA. Theresulting siRNA can then inhibit expression of the target gene. For adiagram of the process, see FIG. 18.

Use of dsRNA for RNAi Analysis

Long dsRNA duplexes can be used directly for RNAi analysis in organismsand systems lacking the interferon response, including insects(Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999), insectcell lines (Caplen et al., 2000), C. elegans (Fire et al., 1998),trypanosomes (Ngo et al., 1998), some mammalian embryonic cell lines(Billy et al., 2001; Yang et al., 2001), and mouse oocytes andpreimplantation embryos (Svoboda et al., 2000; Wianny andZernicka-Goetz, 2000).

Long dsRNA duplexes cannot be used directly for RNAi analysis in mostsomatic mammalian cell lines. This is because introduction of dsRNA intothese cell lines induces a non-specific, interferon-mediated responseresulting in shutdown of translation and initiation of cellularapoptosis (Kaufman, 1999). To perform RNAi analysis in mammalian celllines, long dsRNA should first be cleaved into 21-23 nucleotide siRNAduplexes. This cleavage process may be performed in vitro usingrecombinant Dicer enzyme such as is provided in the BLOCK-iT™ Dicer RNAiTransfection Kit or the BLOCK-iT™ Complete Dicer RNAi Kit. For moreinformation, refer the BLOCK-iT™ Dicer RNAi Kits manual.

Experimental Outline

The table below describes the major desired steps to generate a dsRNAusing the BLOCK-iT™ RNAi TOPO® Transcription Kit. Step Action 1 Produceyour PCR product using Taq polymerase or Platinum ® Taq DNA polymerase.2 Verify the integrity and concentration of your PCR product. 3 Performthe TOPO ® Linking reaction to link your PCR product to the BLOCK-iT ™T7-TOPO ® Linker. 4 Amplify the TOPO ® Linked PCR product using theappropriate primers to produce sense and antisense linear DNA templates.5 Use each linear DNA template in an RNA transcription reaction toproduce sense and antisense RNA transcripts. 6 Purify sense andantisense RNA transcripts. 7 Quantitate the yield of each purified ssRNAobtained, and anneal equal amounts of sense and antisense ssRNA togenerate dsRNA.

Methods

Designing PCR Primers

To use BLOCK-iT™ RNAi TOPO® Transcription Kit, you will first need todesign PCR primers to amplify your sequence of interest. Guidelines tochoose the target sequence and to design PCR primers are provided below.

Choosing the Target Sequence

When performing RNAi analysis, your choice of target sequence cansignificantly affect the degree of gene knockdown observed. In addition,the size of the target sequence and the resulting dsRNA can affect thetranscription efficiency and thus the yield of dsRNA produced. Considerthe following factors when choosing your target sequence.

-   1. Select the target sequence that covers a reasonable portion of    the gene of interest and that does not contain regions of strong    homology with other genes.-   2. Limit the size of the target sequence. Although smaller or larger    target sequences are possible, we recommend limiting the initial    target sequence to a size range of 500 bp to 1 kb for the following    reasons.    -   (a) This balances the risk of including regions of strong        homology between the target gene and other genes that could        result in non-specific off-target effects during RNAi analysis        with the benefits of using a more complex pool of siRNA.    -   (b) When producing sense and antisense transcripts of the target        template, the highest transcription efficiencies are obtained        with transcripts in the 500 bp to 1 kb size range. Target        templates outside this size range transcribe less efficiently,        resulting in lower yields of dsRNA.    -   (c) If you plan to “dice” the dsRNA to produce d-siRNA for use        in mammalian RNAi analysis, note that dsRNA that are under 1 kb        in size are efficiently diced. Larger dsRNA can be used but        yields may decline as the size increases.

The BLOCK-iT™ Complete Dicer RNAi Kit has been used successfully toknock down gene activity with dsRNA substrates ranging from 150 bp to1.3 kb in size.

Factors to Consider When Designing PCR Primers

Once you have selected an appropriate target sequence, you will need todesign gene-specific primers to amplify your target sequence ofinterest. Consider the following factors when designing gene-specificprimers.

-   1. Make sure that your primers do not contain sequence that is    homologous to other genes.-   2. Once you have linked your primary PCR product to the BLOCK-iT™    T7-TOPO® Linker, you will amplify the resulting linked product using    the BLOCK-iT™ T7 Primer and either your gene-specific forward primer    or gene-specific reverse primer. When designing your gene-specific    PCR primers, make sure that the Tm of each primer is compatible with    the Tm of the BLOCK-iT™ T7 primer (i.e. Tm=62° C.).

FIG. 19 can be used to design appropriate PCR primers to join yoursequence of interest with the BLOCK-iT™ T7-TOPO® Linker. The BLOCK-iT™T7TOPO® Linker is supplied as a double-stranded DNA fragment adaptedwith topoisomerase I.

Features of the BLOCK-iT™ T7-TOPO® Linker:

-   -   The sequence of the T7 promoter is indicated in bold.    -   The transcription start site is indicated by +1.

To obtain consistent and efficient results in the TOPO® Linkingreaction, we recommend using HPLC-purified oligonucleotides to produceyour PCR products. Using a mixture of full-length and non full-lengthprimers to produce your PCR products can reduce the efficiency of TOPO®Linking and result in poor yield of the linear DNA templates aftersecondary amplification.

Do not add 5′ phosphates to your primers for PCR. This will preventTOPO® Linking.

Amplifying Your Sequence of Interest

Once you have decided on a PCR strategy and have synthesized theprimers, you are ready to produce your PCR product.

Choosing a Thermostable DNA Polymerase

To amplify your sequence of interest, use a thermostable DNA polymerasethat generates PCR products with 3′ A-overhangs. We recommend usingPlatinum® Taq polymerase available from Invitrogen. Taq polymerase isalso suitable.

You may use Taq polymerase and proofreading polymerase mixtures togenerate PCR products, however, a certain proportion of your PCRproducts will be blunt-ended. You can add 3′ A-overhangs to your PCRproducts using the method below.

Control Plasmid

We recommend amplifying the control template included with the kit inparallel with your sample. Use the LacZ Forward 2 and the LacZ Reverse 2primers included with the kit to amplify the pcDNA™ 1.2/V5-GW/lacZplasmid. The resulting control PCR product (representing a 1 kb fragmentof the lacZ gene) may then be used as a positive control for subsequentprocedures including TOPO® Linking, transcription, and production ofdsRNA. For a map of pcDNA™ 10.2/V5-GW/lacZ, refer to FIG. 21.

To use the pcDNA™ 1.2/V5-GW/lacZ plasmid as a template foramplification, resuspend the plasmid in 10 μl of sterile water to obtaina final concentration of 1 μg/μl. Dilute as appropriate and use 1-10 ngof plasmid DNA in the PCR reaction.

Materials Needed

You should have the following materials on hand before beginning:

-   -   1. Thermocycler    -   2. Thermostable DNA polymerase (e.g. Platinum® Taq DNA        Polymerase)    -   3. DNA template    -   4. Gene-specific forward and reverse PCR primers (10 μM each)    -   5. 10×PCR Buffer (supplied with the kit, Box 1)    -   6. 40 mM dNTPs (supplied with the kit, Box 1)    -   7. Sterile water (supplied with the kit, Box 1)

Setting Up the PCR Reaction

Use the procedure below to amplify your sequence of interest usingPlatinum® Taq DNA polymerase. Use less DNA if you are using plasmid DNAas a template (1-10 ng) and more DNA if you are using genomic DNA as atemplate (10-100 ng).

If you are using a different thermostable DNA polymerase, reactionconditions may vary.

1. Set up the following 50 μl PCR reaction. DNA Template 1-100 ng 10XPCR Buffer 5 μl 40 mM dNTPs 1 μl PCR Primers (10 μM each) 1 μl eachSterile water add to a final volume of 49.5 μl Platinum ® Taq polymerase(5 U/μl) 0.5 μl Total volume 50 μl

-   2. Use the cycling parameters suitable for your primers and    template. Be sure to include a 7 minute extension at 72° C. after    the last cycle to ensure that all PCR products are full-length and    3′ adenylated.-   3. After cycling, place the tube on ice. Proceed to Checking the PCR    Product, below.

Checking the PCR Product

Analyze 1-5 μl of the PCR reaction using agarose gel electrophoresis toverify the quality and quantity of your PCR product. Check for thefollowing:

-   1. A single discrete band of the expected size corresponding to your    sequence of interest. If you do not obtain a single, discrete band    from your PCR, follow the manufacturer's recommendations or use the    PCR Optimizer™ Kit (Catalog no. K1220-01) from Invitrogen to    optimize your PCR conditions using your DNA polymerase. Other tips    may be found below or in published reference sources (Innis et al.,    1990). Alternatively, you may gel-purify your fragment before    proceeding to TOPO® Linking.-   2. Estimate the concentration of your PCR product. For optimal TOPO®    Linking, the concentration of your PCR should be ≧20 ng/μl. If your    PCR product is too dilute, see Concentrating Dilute PCR Products,    below.

Once you have verified that your PCR product is of the appropriatequality and concentration, proceed to Performing the TOPO® LinkingReaction.

For optimal results, use fresh PCR product in the TOPO® Linkingreaction.

You may store the PCR product at −20° C. for up to 1 week.

Concentrating Dilute PCR Products

If you obtain a single band from PCR, but your PCR product is toodilute, you may purify and concentrate the PCR product before proceedingto the TOPO® Linking reaction. A procedure to purify and concentrate PCRproducts is provided below.

Performing the TOPO® Linking Reaction

Introduction

Once you have produced your PCR product, you will use TOPO® Linking tojoin the PCR product to the BLOCK-iT™ T7-TOPO® Linker. Before performingthe TOPO® Linking reaction, you should have everything you need set upand ready to use to ensure that you obtain the best results. If you haveproduced the control PCR product and this is the first time you haveperformed TOPO® Linking, we recommend performing the control TOPO®Linking reaction below in parallel with your samples.

Materials Needed

Have the following reagents on hand before beginning:

-   -   1. Your primary PCR product (≧20 ng/μl)    -   2. BLOCK-iT® T7-TOPO® Linker (supplied with the kit, Box 1; keep        at −20° C. until use)    -   3. Salt Solution (supplied with the kit; Box 1)    -   4. Sterile Water (supplied with the kit; Box 1)    -   5. 37° C. water bath

TOPO® Linking Procedure

Follow the procedure below to perform the TOPO® Linking reaction.

1. Set up a 6 μl TOPO® Linking reaction using the following reagents inthe order given. Your PCR product (≧20 ng/μl) 1 μl Salt Solution 1 μlSterile water 3 μl BLOCK-iT ™ T7-TOPO ® Linker 1 μl Total volume 6 μl

-   2. Mix reaction gently and incubate for 15 minutes at 37° C. Do not    incubate the reaction for longer than 15 minutes as this may    negatively affect TOPO® Linking.-   3. Place the reaction on ice and proceed directly to Performing    Secondary Amplification.    You may store the TOPO® Linking reaction at −20° C. overnight, if    desired.

Performing Secondary Amplification Reactions

Introduction

Once you have performed the TOPO® Linking reaction, you will use thisreaction mixture in two PCR reactions with the appropriate PCR primersto produce sense and antisense linear DNA templates. Guidelines toperform secondary amplification are provided in this section.

Thermostable DNA Polymerase

You may use any thermostable DNA polymerase to produce sense andantisense linear DNA templates. We generally use the same thermostableDNA polymerase to perform secondary amplification as we use to generatethe primary PCR product (i.e. Platinum® Taq DNA Polymerase).

PCR Primers

To produce sense and antisense linear DNA templates, you will performtwo amplification reactions using the TOPO® Linking reaction and theappropriate primers (see table below). For gene-specific PCR primers,use the primers that you used to produce your primary PCR product. TheBLOCK-iT™ T7 Primer is supplied with the kit. Sense Template AntisenseTemplate BLOCK-iT ™ T7 Primer BLOCK-iT ™ T7 Primer Gene-specific reverseprimer Gene-specific forward primer

General Guidelines

When amplifying the TOPO® Linked PCR product, we recommend thefollowing:

-   -   1. Perform the PCR reaction in a total volume of 50 μl.    -   2. Use 1 μl of the TOPO® Linking reaction as the DNA template.    -   3. If you use the same thermostable DNA polymerase to perform        secondary amplification as was used to generate the primary PCR        product, you may generally use similar cycling conditions.        However, because you are using different PCR primers, you may        need to adjust the cycling conditions.

Materials Needed

You should have the following materials on hand before beginning:

-   -   1. Thermocycler    -   2. Thermostable DNA polymerase (e.g. Platinum® Taq DNA        Polymerase)    -   3. TOPO® Linking reaction (from Step 3)    -   4. Gene-specific forward and reverse primers (10 D]M each)    -   5. BLOCK-iT™ T7 Primer (supplied with the kit, Box 1)    -   6. 10×PCR Buffer (supplied with the kit, Box 1)    -   7. 40 mM dNTPs (supplied with the kit, Box 1)    -   8. Sterile water (supplied with the kit, Box 1)

Setting Up the Secondary PCR Reactions

Use the procedure below to amplify the TOPO® Linked PCR product usingPlatinum® Taq DNA polymerase. If you are using a different thermostableDNA polymerase, reaction conditions may vary.

1. Set up the following 50 μl PCR reactions: Antisense Reagent SenseTemplate Template 10X PCR Buffer 5 μl 5 μl 40 mM dNTPs 1 μl 1 μlBLOCK-iT ™ T7 Primer (75 ng/μl) 1 μl 1 μl Gene-specific forward primer(10 μM) — 1 μl Gene-specific reverse primer (10 μM) 1 μl — Sterile water40.5 μl 40.5 μl TOPO ® Linking reaction 1 μl 1 μl Platinum ® TaqPolymerase (5 U/μl) 0.5 μl 0.5 μl Total volume 50 μl 50 μl

-   2. Use the cycling parameters suitable for your primers and    template. Be sure to include a 7 minute extension at 72° C. after    the last cycle to ensure that all PCR products are full-length.-   3. After cycling, place the tube on ice. Proceed to Checking the PCR    Products, below.

Checking the PCR Products

Analyze 1-5 μl of each PCR reaction using agarose gel electrophoresis toverify the quality and quantity of your PCR product. Check for thefollowing:

-   -   1. A single discrete band of the expected size corresponding to        your linked linear DNA template.    -   2. You may see some minor background bands. These are generally        due to smaller PCR products that were in the primary PCR        reaction and should not affect the efficiency of the        transcription reaction.

3. Estimate the concentration of each PCR product. For optimaltranscription efficiency, the concentration of each PCR product shouldbe ≧25 ng/μl. If your PCR product(s) is too dilute, you may increase thenumber of cycles of the amplification reaction or use the procedureprovided below to purify and concentrate your PCR product.

Once you have verified that your PCR products are of the appropriatequality and concentration, proceed to Performing the RNA TranscriptionReaction.

Storing the PCR Products

For optimal results, use fresh PCR products in the RNA transcriptionreaction. You may store the PCR products at −20° C. for up to 1 month,if desired.

Performing the RNA Transcription Reactions

Once you have produced the sense and antisense DNA templates of yourtarget sequence, you will perform two transcription reactions using thereagents supplied in the RNA Transcription Kit (Box 2) to generate senseand antisense transcripts.

Amount of DNA Template to Use

For each RNA transcription reaction, you will need 250 ng to 1 μg ofyour DNA template. For best results, make sure that the concentration ofyour sense and antisense DNA templates is ≧25 ng/μl.

Positive Control

If you have performed the control reactions described, we recommendusing the resulting sense and antisense lacZ templates as controls inthe RNA transcription, purification, and annealing procedures. Once youhave produced control lacZ dsRNA, you may:

-   -   Use this dsRNA as a negative control for non-specific,        off-target effects in your RNAi studies.    -   Include the lacZ dsRNA in a dicing reaction (refer to the        BLOCK-iT™ Dicer RNAi Kits manual for instructions), then use the        resulting lacZ d-siRNA as a positive control for RNAi in        mammalian cells. Co-transfect the lacZ d-siRNA and the pcDNA™        1.2/V5-GW/lacZ plasmid into mammalian cells and assay for        knockdown oft-galactosidase expression.

When performing the RNA transcription reaction and all subsequentprocedures, take precautions to avoid RNase contamination.

-   -   Use RNase-free, sterile pipette tips and supplies for all        manipulations.    -   Use DEPC-treated solutions as necessary.    -   Wear gloves when handling reagents and solutions and when        setting up the transcription reaction.

Materials Needed

You should have the following materials on hand before beginning:

-   -   1. Sense and antisense DNA templates (from the Secondary        Amplification reactions, Step 3; ≧25 ng/μl each)    -   2. RNase-Free Water (supplied with the kit, Box 2)    -   3. 75 mM NTPs (supplied with the kit, Box 2)    -   4. 10× Transcription Buffer (supplied with the kit, Box 2; keep        on ice until use)    -   5. BLOCK-iT™ T7 Enzyme Mix (supplied with the kit, Box 2; keep        at −20° C. until use)    -   6. DNase I (supplied with the kit, Box 2)    -   7. RNase-free supplies (e.g. microcentrifuge tubes and pipette        tips)    -   8. 37° C. water bath

Guidelines to Set Up the Transcription Reactions

Follow the guidelines below when setting up the transcription reactions.

-   -   a) Set up the transcription reaction at room temperature. Do not        set up the reaction on ice as components in the transcription        buffer may precipitate the DNA template.    -   b) keep the 10× Transcription Buffer on ice; do not thaw until        immediately before use.    -   c) Upon thawing the 10× Transcription Buffer, you may notice        some precipitate in the bottom of the tube. Warm the buffer to        37° C. and vortex briefly to allow the precipitate to go back        into solution.    -   d) When setting up the transcription reaction, add the        components to the microcentrifuge tube exactly in the order        stated. Add the 10× Transcription Buffer to the mixture directly        before adding the BLOCK-iT™ T7 Enzyme Mix, and mix immediately        to avoid precipitation of the template. After use, return the        10× Transcription Buffer and the BLOCK-iT™ T7 Enzyme Mix to −20°        C.

RNA Transcription Procedure

Use the procedure below to synthesize transcripts from your DNAtemplate. Remember that for each gene, you will generate sense andantisense transcripts using the sense and antisense DNA templates,respectively. Be sure to use RNase-free supplies and wear gloves toprevent RNase contamination.

If you wish to include a negative control, set up the transcriptionreaction as described below, except omit the DNA template.

1. For each sample, add the following components exactly in the orderstated to a 0.5 ml sterile, microcentrifuge tube at room temperature andmix. The amount of RNase-free water added will depend on theconcentration of your DNA template. Reagents Amount RNase-Free Water upto 21 μl 75 mM NTPs 8 μl DNA template (250 ng-1 μg) 1-10 μl 10XTranscription Buffer 4 μl BLOCK-iT ™ T7 Enzyme Mix 6 μl Total volume 40μl

-   2. Incubate the reaction at 37° C. for 2 hours.

The length of the RNA transcription reaction can be extended up to 6hours. Most of the transcripts are produced within the first 2 hours,but yields can be increased with longer incubation.

-   3. Add 2 μl of DNase I to each reaction. Incubate for 15 minutes at    37° C.-   4. Proceed to Purifying RNA Transcripts.    You may store the RNA transcription reactions at −20° C. overnight    before purification, if desired.

Purifying RNA Transcripts

This section provides guidelines and instructions to purify thesingle-stranded RNA transcripts (ssRNA) produced in the RNAtranscription reaction. Use the BLOCK-iT™ RNA Purification reagents (Box3) supplied with the kit. Remember that for each gene, you will perform2 purification reactions to purify sense and antisense RNA transcripts.

Experimental Outline

To purify RNA transcripts, you will:

-   1. Add RNA Binding Buffer and ethanol to the transcription reaction    to denature the proteins and to enable the ssRNA to bind to the    column.-   2. Add the sample to an RNA spin cartridge. The ssRNA binds to the    silica-based membrane in the cartridge, and the digested DNA, free    NTPs, and denatured proteins flow through the cartridge.-   3. Wash the membrane-bound ssRNA to eliminate residual RNA Binding    Buffer and any remaining impurities.-   4. Elute the ssRNA from the RNA spin cartridge with water.

Advance Preparation

Before using the BLOCK-iT™ RNA Purification reagents for the first time,add 10 ml of 100% ethanol to the entire amount of 5×RNA Wash Buffer togenerate a 1×RNA Wash Buffer (total volume=12.5 ml). Place a check inthe box on the 5×RNA Wash Buffer label to indicate that the ethanol wasadded. Store the 1×RNA Wash Buffer at room temperature.

The RNA Binding Buffer contains guanidine isothiocyanate. This chemicalis harmful if it comes in contact with the skin or is inhaled orswallowed. Always wear a laboratory coat, disposable gloves, and goggleswhen handling solutions containing this chemical.

Do not add bleach or acidic solutions directly to solutions containingguanidine isothiocyanate or sample preparation waste. Guanidineisothiocyanate forms reactive compounds and toxic gases when mixed withbleach or acids.

Materials Needed

Have the following materials on hand before beginning:

-   -   1. RNA transcription reactions (from Step 4; for each gene, you        should have a sense transcription reaction and an antisense        transcription reaction)    -   2. RNA Binding Buffer (supplied with the kit, Box 3)    -   3. β-mercaptoethanol    -   4. 100% ethanol    -   5. RNA spin cartridges (supplied with the kit, Box 3; one for        each sample)    -   6. 1×RNA Wash Buffer (see Advance Preparation, above)    -   7. RNase-Free Water (supplied with the kit, Box 3)    -   8. RNA Recovery Tubes (supplied with the kit, Box 3; one for        each sample)    -   9. 50×RNA Annealing Buffer (supplied with the kit, Box 3)

ssRNA Purification Procedure

Use this procedure to purify ssRNA produced in the transcriptionreaction, Step 4.

Immediately before beginning, remove the amount of RNA Binding Bufferneeded and add β-mercaptoethanol to a final concentration of 1% (v/v).Use fresh and discard any unused solution.

-   1. To each RNA transcription reaction (˜40 μl volume), add 160 μl of    RNA Binding Buffer containing 1% (v/v) β-mercaptoethanol followed by    100 μl of 100% ethanol to obtain a final volume of 300 μl. Mix well    by pipetting up and down 5 times.-   2. Apply the sample (˜300 μl) to the RNA Spin Cartridge. Centrifuge    at 14,000×g for 15 seconds at room temperature. Discard the    flow-through.-   3. Add 500 μl of 1X RNA Wash Buffer to the RNA Spin Cartridge    containing bound ssRNA. Centrifuge at 14,000×g for 15 seconds at    room temperature. Discard the flow-through.-   4. Repeat the wash step (Step 3, above).-   5. Centrifuge the RNA Spin Cartridge at 14,000×g for 1 minute at    room temperature to remove residual 1×RNA Wash Buffer from the    cartridge and to dry the membrane.-   6. Remove the RNA Spin Cartridge from the Wash Tube, and place it in    an RNA Recovery Tube.-   7. Add 40 μl of RNase-Free Water to the RNA Spin Cartridge. Let    stand at room temperature for 1 minute, then centrifuge the RNA Spin    Cartridge at 14,000×g for 2 minutes at room temperature to elute the    ssRNA.-   8. Add 40 μl of RNase-Free Water to the RNA Spin Cartridge and    repeat Step 7, eluting the ssRNA into the same RNA Recovery Tube.    The total volume of eluted ssRNA is 80 μl.-   9. Depending on your downstream application, perform the following:-   (a) If you plan to use the purified ssRNA to generate dsRNA for use    in RNAi studies, add 1.4 μl of 50×RNA Annealing Buffer to the eluate    to obtain a final concentration of 1×RNA Annealing Buffer. Proceed    to Determining the RNA Concentration, or to Step 10.-   (b) If you plan to use the purified ssRNA for applications such as    Northern analysis, proceed to Step 10.-   10. Store the purified ssRNA at −80° C.

Determining the ssRNA Purity and Concentration

Follow the guidelines below to determine the purity and concentration ofyour purified ssRNA.

-   1 Dilute an aliquot of the purified ssRNA 100-fold into 1×RNA    Annealing Buffer in a total volume appropriate for your quartz    cuvette and spectrophotometer.-   2. Measure OD at A260 and A280 in a spectrophotometer. Blank the    sample against 1×RNA Annealing Buffer.-   3. Calculate the concentration of the ssRNA by using the following    equation:    ssRNA concentration (μg/ml)=A260×Dilution factor (100)×40 μg/ml.-   4. Calculate the yield of the ssRNA by using the following equation:    ssRNA yield (μg)=ssRNA concentration (μg/ml)×volume of ssRNA (ml)-   5. Evaluate the purity of the purified ssRNA by determining the    A260/A280 ratio. For optimal purity, the A260/A280 ratio should    range from 1.9-2.2.

How Much ssRNA to Expect

The typical yield of purified ssRNA obtained from a 1 kb DNA templateranges from 50-80 μg in a 40 μl transcription reaction. However, yieldsmay vary depending on the size of the DNA template and its sequence.Generally, ssRNA yields are lower for DNA templates smaller than 500 bpor larger than 1 kb.

After purification, we recommend saving an aliquot of your sense andantisense ssRNA samples for gel analysis. We generally verify theintegrity of the dsRNA sample (after annealing) and compare it to thesense and antisense ssRNA samples using agarose or polyacrylamide gelelectrophoresis.

If you wish to verify the integrity of your sense and antisense ssRNAsamples before annealing, we suggest running a small aliquot of eachsample on a 6% Novex® TBE-Urea Gel (Invitrogen, Catalog no. EC68652BOX),and including the 0.16-1.77 kb RNA Ladder (Invitrogen, Catalog no.15623-010) as a molecular weight standard.

Generating dsRNA

To generate dsRNA, you will anneal equal amounts of the purified senseand antisense transcripts of your gene of interest (from ssRNAPurification Procedure, Step 8). Guidelines and instructions areprovided below.

Amount of ssRNA to Anneal

You may anneal any amount of sense and antisense transcripts to generatedsRNA; however, use equal amounts of each transcript for optimalresults. We generally anneal 50-80 μg of ssRNA to generate 100-160 μg ofdsRNA, respectively (e.g. annealing 50 μg of sense transcripts and 50 μgof antisense transcripts results in 100 μg of dsRNA). You may assumethat the annealing step is nearly 100% efficient. You will need to knowthe concentration of each ssRNA before beginning.

Materials Needed

Have the following materials on hand before beginning.

-   -   1. Purified sense transcripts of your gene of interest    -   2. Purified antisense transcripts of your gene of interest    -   3. 50×RNA Annealing Buffer (supplied with the kit, Box 3)    -   4. 0.5 ml sterile, RNase-free microcentrifuge tube    -   5. 500 ml glass beaker

Annealing Procedure

Use the procedure below to anneal sense and antisense transcripts togenerate dsRNA. Remember to use RNase-free supplies and wear gloves toprevent RNase contamination.

-   1. In a sterile, RNase-free microcentrifuge tube, mix equal amounts    of purified sense and antisense transcripts. Place the tube on ice.-   2. Heat approximately 250 ml of water to boiling in a 500 ml glass    beaker.-   3. Remove the beaker of water from the hot plate or microwave and    set on your laboratory bench.-   4. Place the tube containing the mixture of sense and antisense    transcripts in a tube float or a rack in the glass beaker.-   5. Allow the water to cool to room temperature for 1-1.5 hours. The    ssRNAs will anneal during this time.-   6. Remove a small aliquot of dsRNA and analyze by agarose or    polyacrylamide gel electrophoresis to check the quality of your    dsRNA.-   7. Store the dsRNA at −80° C. Depending on the amount of dsRNA    produced and your downstream application, you may want to aliquot    the dsRNA before storage at −80° C.

When using the dsRNA, avoid repeated freezing and thawing as dsRNA candegrade with each freeze/thaw cycle.

Alternative Annealing Procedure

If you want to generate dsRNA more quickly, use the alternativeannealing procedure below. Note however, that this method is lessefficient and will result in lower yields of dsRNA than theslow-annealing method described above.

-   1. In a sterile, RNase-free microcentrifuge tube, mix equal amounts    of purified sense and antisense transcripts.-   2. Place the tube in a 75° C. heat block for 5 minutes.-   3. Remove the tube from the heat block and place in a rack at room    temperature for 5 minutes. The ssRNAs will anneal during this time.-   4. Remove a small aliquot of dsRNA and analyze by agarose or    polyacrylamide gel electrophoresis to check the quality of your    dsRNA (see below).-   5. Store the dsRNA at −80° C. Depending on the amount of dsRNA    produced and your downstream application, you may want to aliquot    the dsRNA before storage at −80° C.

When using the dsRNA, avoid repeated freezing and thawing as dsRNA candegrade with each freeze/thaw cycle.

Checking the Integrity of dsRNA

You may verify the integrity of your dsRNA using agarose orpolyacrylamide gel electrophoresis, if desired. We suggest running asmall aliquot of your annealing reaction (equivalent to 100-200 ng ofdsRNA) on the appropriate gel and comparing it to an aliquot (100-200ng) of your starting sense and antisense ssRNA. Be sure to include anappropriate molecular weight standard. We generally use the followinggels and molecular weight standard:

-   -   Agarose gel: 1.2% agarose-TAE gel    -   Polyacrylamide gel: 6% Novex® TBE Gel (Invitrogen, Catalog no.        EC6265BOX)    -   Molecular weight standard: 0.16-1.77 kb RNA Ladder (Invitrogen,        Catalog no. 15623-10)

What You Should See

When analyzing the annealing reaction (see above) using gelelectrophoresis, we generally observe a predominant band correspondingto the dsRNA (see FIG. 20). If you have used one of the recommendedannealing procedures (see above), no ssRNA molecules should be detected.

A high molecular weight smear is often visible in the annealed samples.This generally due to branched annealing that occurs when multipleoverlapping ssRNA anneal to each other. These products can be diced invitro or in vivo to generate siRNA.

Example of Expected Results

In this experiment, dsRNA representing a 730 bp region of the greenfluorescent protein (GFP) gene and a 1 kb region of the luciferase genewere generated using the reagents supplied in the kit and following therecommended protocols in the manual. One microgram of each dsRNA wasanalyzed on a 1.2% agarose-TAE gel and compared to 0.5 μg of eachcorresponding purified sense and antisense ssRNA (non-denatured).

Results are shown in FIG. 20: The annealed GFP (lane 4) and luciferase(lane 7) dsRNA samples both show a predominant band that differs in sizefrom each component sense and antisense ssRNA. No ssRNA is visible inthe annealed sample. A high molecular weight smear due to branchedannealing products is also visible in the annealed samples (lanes 4 and7).

In some cases, multiple bands due to secondary structure are observed inthe ssRNA samples (e.g., lanes 5 and 6). This is a result of analysis onnon-denaturing agarose gels.

What to Do Next

Once you have obtained dsRNA, you have the following options:

-   1. Use the dsRNA directly to perform RNAi studies in invertebrate    systems. Depending on the invertebrate system chosen (e.g. C.    elegans, Drosophila, trypanosomes), multiple methods may exist to    introduce the dsRNA into the organism or cell line of choice    including injection, soaking in media containing dsRNA, or    transfection. Choose the method best suited for your invertebrate    system.-   2. Use the dsRNA in an in vitro reaction with the Dicer enzyme to    generate d-siRNA. The resulting d-siRNA may then be transfected into    mammalian cells for RNAi studies. For optimized reagents and    protocols to generate highly pure d-siRNA from a dsRNA substrate    using recombinant human Dicer enzyme, and to efficiently transfect    the d-siRNA into a mammalian cell line of interest using    Lipofectamine™ 2000 Reagent, we recommend using the BLOCK-iT™ Dicer    RNAi Transfection Kit (Catalog no. K3600-01) or the BLOCK-iT™    Complete Dicer RNAi Kit (Catalog no. K3650-01) available from    Invitrogen. For detailed instructions to perform the dicing and    transfection reactions, refer to the BLOCK-iT™ Dicer RNAi Kits    manual.

Troubleshooting

Review the information in this section to troubleshoot theamplification, TOPO® Linking, transcription, and purificationprocedures.

Amplifying the Gene of Interest

The table below lists some potential problems and possible solutionsthat may help you troubleshoot your amplification reactions. ProblemReason Solution No PCR Poor quality of Prepare new template DNA andverify product DNA template the integrity of the DNA beforeamplification. Poor quality PCR Amplify the control vector using thereagents or inactive primers supplied with the kit and the thermostableDNA protocol above. If no PCR product is polymerase produced, use freshPCR reagents and thermostable DNA polymerase. Suboptimal PCR Check theT_(m) of the PCR primers conditions and adjust your cycling conditions.Optimize PCR conditions. Refer to the manufacturer's recommendations foryour polymerase. Low Suboptimal PCR Optimize PCR conditions. Refer tothe yield of conditions manufacturer's recommendations for PCR yourpolymerase. product Used old DNA Use fresh thermostable DNA polymerasepolymerase. Not enough PCR Increase the number of PCR cycles. cyclesperformed Multiple Suboptimal cycling Optimize PCR conditions. Refer tothe non- conditions manufacturer's recommendations for specific yourpolymerase. bands or DNA template Prepare new template DNA and verifysmearing contaminated the integrity of the DNA before observed withother DNA amplification. on Poor quality PCR Use HPLC-purified primersto produce agarose primers your PCR product. gel

TOPO® Linking and Secondary Amplification

The table below lists some potential problems and possible solutionsthat you may use to help you troubleshoot the TOPO® Linking reaction andthe secondary amplification reactions. Problem Reason Solution No linearDNA Inefficient TOPO ® Linking Do not incubate the template(s) of theIncubated the TOPO ® TOPO ® Linking reaction at expected size Linkingreaction at 37° C. for 37° C. for longer than obtained too long 15minutes. Use Taq polymerase Used a proofreading (e.g. Platinum ® Taq) topolymerase to generate the generate the primary PCR primary PCR productproduct. Alternatively, add 3′ A-overhangs to the PCR product (seeprocedure above). Poor quality PCR reagents or Use fresh PCR reagentsand inactive thermostable DNA thermostable DNA polymerase polymerase forthe secondary amplification reactions. Primers used to produce the Donot add 5′ phosphates primary PCR product to the primers used tocontained 5′ phosphates produce the primary PCR product. TOPO ® Linkingreaction For optimal results, stored incorrectly perform secondaryamplification reactions directly after TOPO ® Linking. If desired, storethe TOPO ® Linking reaction at −20° C. overnight. Low yield of linearInefficient TOPO ® Linking Purify and concentrate DNA template PrimaryPCR product was the PCR product using the obtained too dilute procedureabove. Primary PCR product was For optimal results, use not fresh freshPCR product in the Taq polymerase and TOPO ® Linking reaction.proofreading polymerase Use Taq polymerase to mixture used to generategenerate the primary PCR primary PCR product product or use theprocedure above to add 3′ A-over-hangs to the PCR product prior toTOPO ® Linking. Annealing temperature was Check the T_(m)s of your PCRtoo high primers. Reduce the annealing temperature. T_(m) of thegene-specific Re-design the gene-specific primer(s) not compatible withprimer(s), making sure that the T_(m) of the BLOCK-iT ™ T7 the T_(m) ofeach primer is Primer compatible with the T_(m) of the BLOCK-iT ™ T7Primer. Not enough PCR cycles Increase the number of PCR performedcycles.

Transcribing and Purifying ssRNA

The table below lists some potential problems and possible solutionsthat may help you troubleshoot the transcription and purification steps.Problem Reason Solution Low ssRNA yield No ethanol or RNA Binding AddRNA Binding Buffer Buffer added to the sample containing 1% (v/v) β-mercaptoethanol followed by 100% ethanol to the sample (see ssRNAPurification Procedure, Step 1, above). Linear DNA template too Purifyand concentrate the dilute linear DNA template using the procedure onabove. Extend the incubation time of the transcription reaction up to 6hours at 37° C. Transcription reaction not Extend the incubation time ofincubated long enough the transcription reaction up to 6 hours at 37° C.Eluted ssRNA from the Elute ssRNA from the RNA Spin RNA Spin Cartridgeusing Cartridge using RNase-free buffer, not water water. Concentrationof ssRNA Dilute sample in 1X RNA incorrectly determined Annealing Bufferfor Sample diluted into spectrophotometry. water for Blank sampleagainst 1X spectrophotometry RNA Annealing Buffer. Sample blankedagainst water No ssRNA Sample contaminated with Use RNase-free reagentsand obtained RNase supplies. Wear gloves when handling RNA-containingsamples. Gene-specific primers used Use the BLOCK-iT ™ T7 Primer toamplify TOPO ® Linked and the gene-specific forward or products, not theBLOCK- reverse primer in the secondary iT ™ T7 Primer amplificationreaction to generate sense and antisense DNA templates, respectively.Forgot to add ethanol to the Add 10 ml of ethanol to the 5X 5X RNA WashBuffer RNA Wash Buffer (2.5 ml) to obtain a 1X RNA Wash Buffer. Volumeof eluted RNA Spin Cartridge Centrifuge RNA Spin Cartridge ssRNA is >80μl containing bound ssRNA at 14,000 x g for 1 minute at not centrifugedto remove room temperature to remove residual 1X RNA Wash residual 1XRNA Wash Buffer Buffer and to dry the membrane (see Step 5, above).Contamination of eluted ssRNA with 1X RNA Wash Buffer or otherimpurities can result in inaccurate quantitation of ssRNA, potentialtoxic effects on invertebrate cells, or reduced dicing efficiency.A260/A280 ratio Sample was not washed Wash the RNA Spin Cartridge not inthe 1.9-2.2 with 1X RNA Wash Buffer containing bound ssRNA twice rangewith 1X RNA Wash Buffer (see Steps 3 and 4, above). RNA Spin CartridgeCentrifuge RNA Spin Cartridge containing bound ssRNA at 14,000 x g for 1minute at not centrifuged to remove room temperature to remove residual1X RNA Wash residual 1X RNA Wash Buffer Buffer and to dry the membrane(see Step 5, above).

RNAi Analysis

The table below lists some potential problems and possible solutionsthat may help you troubleshoot your RNAi analysis using dsRNA. ProblemReason Solution Low levels of gene dsRNA was degraded Be sure to storethe dsRNA knockdown dsRNA was not stored in 1X RNA Annealing Buffer.observed in 1X RNA Annealing Buffer Aliquot dsRNA and avoid dsRNA wasfrozen and repeated freeze/thaw cycles. thawed multiple times No geneTarget sequence contains no Select a larger target region or a knockdownactive siRNA different target sequence. observed dsRNA contaminated withUse RNase-free reagents RNase and supplies. Wear gloves when handlingRNA-containing samples. Non-specific gene Target sequence containsSelect a new target knockdown effects strong homology to other genessequence. observed Limit the size range of the target sequence to 1 kb.

Performing the Control Reactions

We recommend performing the following control reactions the first timeyou use the kit to help you evaluate your results. Performing thecontrol reactions involves the following steps:

-   1. Producing a control PCR product using the pcDNA™ 1.2/V5-GW/lacZ    control plasmid and the LacZ Forward 2 and LacZ Reverse 2 primers    supplied with the kit.-   2. Performing a TOPO® Linking reaction with the control PCR product    and the BLOCK-iT® T7-TOPO® Linker.-   3. Performing two secondary amplification reactions with the TOPO®    Linked PCR product to produce sense and antisense control DNA    templates.-   4. Using the control DNA templates in transcription reactions to    generate sense and antisense RNA transcripts.-   5. Purifying the sense and antisense RNA transcripts, and annealing    the ssRNAs to produce control dsRNA.

Producing the Control PCR Product

Use this procedure to amplify the pcDNA™ 1.2/V5-GW/lacZ control plasmidusing Platinum® Taq polymerase. If you are using another thermostableDNA polymerase, follow the manufacturer's instructions to set up the PCRreaction.

1. To produce the 1 kb control PCR product, set up the following 50 μlPCR: pcDNA ™ 1.2/V5-GW/lacZ (10 ng/μl) 1 μl 10X PCR Buffer 5 μl 40 mMdNTPs 1 μl LacZ forward 2 primer (65 ng/μl) 1 μl LacZ reverse 2 primer(65 ng/μl) 1 μl Sterile Water 40.5 μl   Platinum ® Taq Polymerase (5U/μl) 0.5 μl   Total Volume 50 μl 

2. Amplify using the following cycling parameters: Step Time TemperatureCycles Initial Denaturation  2 minutes 94° C. 1X Denaturation 15 seconds94° C. 30X  Annealing 30 seconds 55° C. Extension  1 minute 72° C. FinalExtension  7 minutes 72° C. 1X

-   3. Remove 1-5 μl from the reaction and analyze by agarose gel    electrophoresis. A discrete 1 kb band should be visible.

Control TOPO® Linking Reaction

Using the control PCR product produced in Step 3, above and theBLOCK-iT™ T7-TOPO® Linker, set up the TOPO® Linking reaction asdescribed below.

1. Set up the following control TOPO® Linking reaction: Control PCRproduct 1 μl Salt Solution 1 μl Sterile water 3 μl BLOCK-iT ™ T7-TOPO ®Linker 1 μl Total volume 6 μl

-   2. Incubate at 37° C. for 15 minutes and place on ice.-   3. Proceed directly to the Secondary Control PCR Reactions, below.

Secondary Control PCR Reactions

Use this procedure to amplify the TOPO® Linked control PCR product usingPlatinum® Taq polymerase to generate sense and antisense control DNAtemplates. If you are using another thermostable DNA polymerase, followthe manufacturer's instructions to set up the PCR reaction.

1. Set up the following 50 μl PCR reactions: Sense Reagent TemplateAntisense Template Control TOPO ® Linking Reaction 1 μl 1 μl 10X PCRBuffer 5 μl 5 μl 40 mM dNTPs 1 μl 1 μl BLOCK-iT ™ T7 Primer (75 ng/μl) 1μl 1 μl LacZ Forward 2 Primer (65 ng/μl) — 1 μl LacZ Reverse 2 Primer(65 ng/μl) 1 μl — Sterile Water 40.5 μl   40.5 μl   Platinum ® TaqPolymerase (5 U/μl) 0.5 μl   0.5 μl   Total volume 50 μl  50 μl 

2. Amplify using the following cycling parameters: Step Time TemperatureCycles Initial Denaturation  2 minutes 94° C. 1X Denaturation 15 seconds94° C. 30X  Annealing 30 seconds 55° C. Extension  1 minute 72° C. FinalExtension  7 minutes 72° C. 1X

-   3. Remove 1-5 μl from the reaction and analyze by agarose gel    electrophoresis. A discrete band of approximately 1 kb should be    visible.

Generating Control dsRNA

Once you have generated the sense and antisense control DNA templates,you may use these templates in transcription reactions to produce senseand antisense control transcripts. After purification, these transcriptsmay then be annealed to produce control dsRNA. Follow the protocolsabove to produce and purify sense and antisense transcripts, and toanneal the purified transcripts to produce dsRNA.

What To Do With the Control dsRNA

The lacZ dsRNA may be used as a control for RNAi analysis in thefollowing ways:

Invertebrate Systems:

-   -   Use as a negative control for non-specific activity in any        invertebrate system.        Mammalian Systems:

For some embryonic stem cell (ES) cell lines in which the CMV promoteris active (e.g. AB2.2), you may use the lacZ dsRNA as a positive controlfor gene knockdown (Yang et al., 2001). Simply introduce the pcDNA™0.2/V5-GW/lacZ reporter plasmid and the lacZ dsRNA into cells and assayfor inhibition of β-galactosidase expression.

Alternatively, you may use the lacZ dsRNA in an Invitrogen BLOCK-iT™Dicer RNAi Transfection Kit as a substrate to produce diced shortinterfering RNA (d-siRNA). The lacZ d-siRNA may then be used as anegative control for non-specific activity in the mammalian cell line ofinterest or as a positive control for knockdown of β-galactosidaseexpression from the pcDNA™ 1.2/V5-GW/lacZ reporter plasmid. For detailedinstructions to produce d-siRNA, refer to the BLOCK-iT™ Dicer RNAi Kitsmanual.

Gel Purifying PCR Products

Smearing, multiple banding, primer-dimer artifacts, or large PCRproducts (>1 kb) may necessitate gel purification. If you intend topurify your PCR product, be extremely careful to remove all sources ofnuclease contamination. There are many protocols to isolate DNAfragments or remove oligonucleotides. Refer to Current Protocols inMolecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most commonprotocols. Two simple protocols are provided below.

Using the S.N.A.P.™ Gel Purification Kit

The S.N.A.P.™ Gel Purification Kit (Catalog no. K1999-25) allows you torapidly purify PCR products from regular agarose gels.

-   1. Electrophorese amplification reaction on a 1 to 5% regular TAE    agarose gel. Do not use TBE to prepare agarose gels. Borate will    interfere with the sodium iodide step, below.-   2. Cut out the gel slice containing the PCR product and melt it at    65° C. in 2 volumes of 6 M sodium iodide solution. Add 1.5 volumes    of Binding Buffer.-   3. Load solution (no more than 1 ml at a time) from Step 3 onto a    S.N.A.P.™ column. Centrifuge 1 minute at 3000×g in a microcentrifuge    and discard the supernatant.-   4. If you have solution remaining from Step 3, repeat Step 4.-   5. Add 900 μl of the Final Wash Buffer.-   6. Centrifuge 1 minute at full speed in a microcentrifuge and    discard the flow-through.-   7. Repeat Step 7.-   8. Elute the purified PCR product in 30 μl of sterile water. Use 1    μl for the TOPO® Linking reaction and proceed as described above.

Quick S.N.A.P.™ Method

An even easier method is to simply cut out the gel slice containing yourPCR product, place it on top of the S.N.A.P.™ column bed, and centrifugeat full speed for 10 seconds. Use 1-2 μl of the flow-through in theTOPO® Linking reaction. Be sure to make the gel slice as small aspossible for best results.

Adding 3′ A-Overhangs Post-Amplification

Direct TOPO® Linking of DNA amplified by proofreading polymerases withthe BLOCK-iT™ T7-TOPO® Linker is difficult because of very low TOPO®Linking efficiencies. These low efficiencies are caused by the 3′ to 5′exonuclease activity associated with proofreading polymerases whichremoves the 3′ A-overhangs necessary for TA Cloning®. A simple method isprovided below to clone these blunt-ended fragments.

Before Starting

You will need the following items:

-   -   1. Taq polymerase    -   2. A heat block equilibrated to 72° C.    -   3. Phenol-chloroform (optional)    -   4. 3 M sodium acetate (optional)    -   5. 100% ethanol (optional)    -   6. 80% ethanol (optional)    -   7. TE buffer (optional)

Procedure

This is just one method for adding 3′ adenines. Other protocols may be

-   1. After amplification with Vent® or Pfu polymerase, place vials on    ice and add 0.7-l unit of Taq polymerase per tube. Mix well. It is    not necessary to change the buffer.-   2. Incubate at 72° C. for 8-10 minutes (do not cycle).-   3. Place the vials on ice. Proceed to TOPO® Linking (see above).

If you plan to store your sample(s) overnight before proceeding withTOPO® Linking, you may want to extract your sample(s) withphenol-chloroform to remove the polymerases. After phenol-chloroformextraction, precipitate the DNA with ethanol and resuspend the DNA in TEbuffer to the starting volume of the amplification reaction.

Purifying and Concentrating PCR Products

If your gene of interest has not amplified efficiently and the yield ofyour PCR product is low, you may use the S.N.A.P.™ MiniPrep Kitavailable from Invitrogen (Catalog no. K1900-25) to rapidly purify andconcentrate the PCR product. Other resin-based purification kits aresuitable.

Materials Needed

You should have the following reagents on hand before beginning:

-   -   1. Isopropanol    -   2. Binding Buffer (supplied with the S.N.A.P.™ MiniPrep Kit)    -   3. Wash Buffer (supplied with the S.N.A.P.™ MiniPrep Kit)    -   4. Final Wash Buffer (supplied with the S.N.A.P.™ MiniPrep Kit)    -   5. Sterile water S.N.A.P.™ MiniPrep columns (supplied with the        S.N.A.P.™ MiniPrep Kit)

Purification Protocol

Follow the protocol below to purify your PCR product using the S.N.A.P.™MiniPrep Kit. The protocol provides instructions to purify PCR productsfrom a 50 μl reaction volume. To purify PCR products from largerreaction volumes (e.g. several PCR reactions pooled together), scale upthe volumes of each buffer accordingly. Details about the components ofthe S.N.A.P.™ MiniPrep Kit can be found in the S.N.A.P.™ MiniPrep Kitmanual.

-   1. Add 150 μl of Binding Buffer to the 50 μl PCR reaction. Mix well    by pipetting up and down.-   2. Add 350 μl of isopropanol. Mix well by vortexing.-   3. Immediately load solution from Step 2 onto a S.N.A.P.™ MiniPrep    column. Centrifuge for 30 seconds at 1000×g in a microcentrifuge and    discard the flow-through.-   4. Add 250 μl of the Wash Buffer and centrifuge for 30 seconds at    1000×g in a microcentrifuge. Discard the flow-through.-   5. Add 450 μl of the Final Wash Buffer and centrifuge for 30 seconds    at 1000×g in a microcentrifuge. Discard the flow-through.-   6. Centrifuge for an additional 30 seconds at full-speed in a    microcentrifuge to dry the column.-   7. Transfer the column to a new collection tube. Add 30 pt of    sterile water to the column. Incubate at room temperature for 1    minute.-   8. Centrifuge for 30 seconds at full-speed in a microcentrifuge to    elute the DNA. Collect the flow-through. Use 1 μl in the TOPO®    Linking reaction (see above).

pcDNA™ 1.2/V5-GW/lacZ (6498 bp) (see FIG. 21) is a control vectorexpressing a C-terminally-tagged β-galactosidase fusion protein underthe control of the human cytomegalovirus (CMV) promoter (Andersson etal., 1989; Boshart et al., 1985; Nelson et al., 1987), and was generatedusing the MultiSite Gateway® Three-Fragment Vector Construction Kitavailable from Invitrogen (Catalog no. 12537-023). Briefly, a MultiSiteGateway® LR recombination reaction was performed with pDEST™ R4-R3 andentry clones containing the CMV promoter, lacZ gene, and V5 epitope andTK polyadenylation signal to generate the pcDNA™ 1.2/V5-GW/lacZ vector.β-galactosidase is expressed as a C-terminal V5 fusion protein with amolecular weight of approximately 119 kDa. The complete sequence ofpcDNA™ 1.2/V5-GW/lacZ is available from Invitrogen.

Product Qualification

This section describes the criteria used to qualify the components ofthe BLOCK-iT™ RNAi TOPO® Transcription Kit.

Functional Qualification

The components of the BLOCK-iT™ RNAi TOPO® Transcription Kit arefunctionally qualified as follows:

-   1. Using the pcDNA™ 1.2/V5-GW/lacZ plasmid and the LacZ Forward 2    and LacZ Reverse 2 primers supplied with the kit, a control PCR    product is generated and TOPO® Linked to the BLOCK-iT™ T7-TOPO®    Linker following the protocols above.-   2. Using the BLOCK-iT™ T7 Primer and the LacZ Forward 2 or LacZ    Reverse 2 primer, two aliquots of the TOPO® Linking reaction are    amplified following the procedure above to generate sense and    antisense DNA templates. An aliquot of each secondary PCR reaction    is analyzed on an agarose gel and compared to an aliquot of the    primary PCR product. The sense and antisense DNA template should    demonstrate a gel shift (1043 bp) when compared to the primary PCR    product (1000 bp).-   3. The sense and antisense DNA templates are transcribed using the    reagents supplied in the kit and following the procedure above. The    sense and antisense transcripts are analyzed on a 6% Novex® TBE-Urea    Gel (Invitrogen, Catalog no. EC68652BOX). The 0.16-1.77 kb RNA    Ladder (Invitrogen, Catalog no. 15623-010) is included as a    molecular weight standard. RNA should be visible in the lanes    containing sense and antisense transcripts, while no RNA should be    observed from a transcription reaction using a template generated    from a PCR product that was not linked to the BLOCK-iT™ T7 Linker.-   4. The sense and antisense transcripts are purified using the    reagents supplied in the kit and following the procedure above.    Following purification, the purified sense and antisense ssRNA are    quantitated using spectrophotometry. Each transcription reaction    should yield at least 60 μg of ssRNA, and the A260/A280 ratio should    be between 1.9 and 2.2-   5. Equal amounts of sense and antisense RNA are annealed following    the procedure above. The dsRNA is analyzed on a 6% Novex® TBE Gel    (Invitrogen, Catalog no. EC6265BOX) with the 0.16-1.77 kb RNA Ladder    included as a molecular weight standard. A gel shift representing    dsRNA should be observed in the annealed sample when compared to    sense or antisense ssRNA.

pcDNA™ 1.2/V5-GW/lacZ Plasmid

The pcDNA™ 1.2/V5-GW/lacZ plasmid is qualified by restriction analysis.Restriction digest should demonstrate the correct banding pattern whenelectrophoresed on an agarose gel.

PCR Primers

The BLOCK-iT™ T7, LacZ Forward 2, and LacZ Reverse 2 primers arefunctionally qualified by performing the control PCR reactions describedon pages above.

REFERENCES

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Struct. Biol. 8, 746-750.

EXAMPLE 4 Small Nucleic Acids Purification System

All catalog numbers provided below correspond to Invitrogen Corporationproducts, Carlsbad, Calif., unless otherwise noted.

Small nucleic acid molecules, especially siRNA, is getting greatattention with function in gene specific knockout or silencing of geneexpression. Recently, many researchers demonstrated that gene specificsiRNA can be generated in vitro via a combination of transcription and aribonuclease enzyme. The digestion of long transcripts is accomplishedwith a ribonuclease called RNase III or Dicer and the digested sample isrequired to be purified from the non-processed template, intermediateand buffer component of enzyme reaction. If residual long dsRNA templateand other intermediates remained in the sample and were transfectedalong with siRNA into cells, it might lead to non-specific response.Thus removal of this residual template and intermediate is required foraccurate functional analysis of the gene specific siRNA. Pre-existingtotal RNA purification systems are not suitable and not designed topurify less than 30 bp nucleic acids and only a size exclusion spincolumn has been utilized to select small size nucleic acid frommixtures.

We have developed a buffer formulation to purify dsRNA that is smallerthan 30 bp using our pre-existing glass fiber filter. Both single-columnand double-column method were developed to purify siRNAs generated usingDicer and RNase III. The purified siRNA can be used to assay cellularfunctional via gene specific knock out without non-specific interferenceby >30 bp dsRNA (complete buffer exchange; eluted in DEPC treated H₂O).This purification procedure can be utilized for other applications suchas linker, aptamer, protein binding domain extraction, etc.

Introduction

Total RNA is composed of three main transcript categories. These areribosomal RNAs (28S, 18S, and 5S in the case of mammalian cells), mRNA,and low molecular weight RNA species such as tRNA, snRNA, and others.The recent discovery and rudimentary elucidation of the mechanism ofaction of RNA interference and the identification of a new regulatoryRNA termed short interfering RNA (siRNA) as well as micro RNA arereceiving increasing attention by the scientific community. Thisincreased interest is based on siRNA's ability to mediatedown-regulation of gene expression by sequence specific, and hence genespecific, degradation of targeted mRNA. The popularity of the siRNAapproach is justified as it has distinct advantages over anti-sensemethods and knockout approaches. It appears that the siRNA approach iscapable of down-regulating gene expression with higher efficiency andefficacy than the antisense approach and offers greater flexibility andease of use compared to knockout approaches.

RNA interference is a cellular defense mechanism where a longdouble-stranded RNA molecule is processed by an endogenous(endo)ribonuclease resulting in the production of small interfering RNAs(siRNAs), which are generally 21 to 23 nucleotides in length. The siRNAmolecules bind to a protein complex, RNA Induced Silencing Complex(RISC), which contains a helicase activity that unwinds siRNA molecules,allowing the anti-sense strand of siRNA to bind to complementary mRNA,thus triggering targeted mRNA degradation by endonucleases or blockingmRNA translation into protein (for a review see Denli and Hannon, 2003,Carrington and Ambros, 2003). In addition, siRNA does not trigger animmune response, because it is a natural cellular mechanism (Sledz et.al., 2003)

Initial attempts of gene specific knockdown using long dsRNA transcriptsfailed in mammalian cells because of activation of protein kinase PKRand 2′,5′-oligoadenylate synthetase that trigger non-specific shutdownof protein synthesis and non-specific degradation of mRNA. Elbashir andco-workers demonstrated that transfection of chemically synthesized21-23 nt dsRNA fragments could specifically suppress gene expressionwithout triggering non-specific gene silencing effects in mammaliancells. However, different suppression levels are often observed withsynthetic short siRNAs as they target a single specific site. Underthese conditions site accessibility becomes an issue as mRNA containinghigh levels of secondary or tertiary structure may preventsiRNA/target/RISC complex formation and affect efficacy of the siRNAused. Thus, multiple double-stranded siRNA molecules, usually 4-5, needto be screened that target different sequences in a targeted mRNA toidentify one siRNA construct with adequate potency for gene suppressionin a given mRNA. Short interfering RNA constructs can also be generatedby transcription in vitro from short DNA templates or by transcriptionin vivo from a transfected DNA construct. However, none of the lattermethods are easily scaled up for multiple gene screens due to high costof oligonucleotides and/or difficulties of target region selection. Anew method was recently developed to generate gene specific functionalsiRNA pools using a combination of RNA transcription followed bydigestion with Dicer enzyme. This method generates multiple functionalsiRNAs from long dsRNA target sequences which correspond to the genetranscript of interest. With this new method, low cost and highlyefficient screening of gene knockdown effects is possible and highthroughput screening of multiple genes can be achieved. However, thelatter methodology requires purification of functional siRNA afterdigestion of long dsRNA substrate with Dicer. Undigested, long dsRNAsubstrates as well as intermediate digestion products longer thanapproximately 30 bp elicit non-specific responses such as non-specificshutdown of translation and initiation of apoptosis (Kaufinan, 1999).Others have used size exclusion columns for purification of functionalsiRNA. However, this purification is not efficient and does not providehigh quality siRNA for transfection.

Our Small Nucleic Acids Purification System provides an efficient meansof purification for functional, diced siRNA and other small dsRNAmolecules. The purification is based on glass fiber purificationtechnology. The small nucleic acids purification system eliminates dsRNAthat exceeds 30 bp in length and selectively and specifically purifiesdsRNA shorter than 30 base pairs. In the case of siRNA, the purifieddsRNA is of high quality, highly functional for transcript specific genesuppression, and exhibits no cell toxicity. Currently, InvitrogenBlock-iT™ Dicer RNAi Kits provide complete Dicer RNAi transfection kit,include RNAi purification kit, Dicer Enzyme kit, Lipofectamine™ 2000Reagent and/or TOPO® transcription Kit, as bundle product. Small NucleicAcids Purification kit is a stand-alone product of the siRNApurification module from Block-iT™ that accommodates not only siRNApurification generated by Dicer and RNase III but also other smallnucleic acids applications. The Small Nucleic Acids Purification Kit, asrelated to the purification of enzymatically-generated siRNA (Dicer &RNaseIII), will generally meet the following criteria: (1) PurifiedsiRNA expected not to contain dsRNA molecules greater than 30 bp inlength, (2) Suppression levels observed with purified siRNA will be thesame or higher than those observed with synthesized siRNA, (3) Recoveryof purified material expected to exceed 80%.

Spin Column Purification Kit Components

-   1. 50 individual spin columns assembled in collection tubes in one    bag-   2. 50 individual recovery tubes in one bag-   3. Binding Buffer (47-6001): 11 mL-   4. 5× Wash Buffer (47-6003): 15 mL, EtOH (95-100%) added by end user-   5. Elution Buffer (47-6002): 3 mL, 1.5 mL EtOH (95-100%) and 1.5 mL    RNase-free water to be added by end user-   6. DEPC water (47-0005): 10 mL-   7. Manual-   8. QRC

The components provided in the kit are sufficient for 50 purificationsusing the single-column protocol, in which a final ethanol precipitationstep in the presence of glycogen as a co-precipitant is desired. Thecomponents provided in the kit are sufficient for 25 purifications whenusing the two-column protocol, in which the second column is used forselective binding of the short target nucleic acids followed by elutionin DEPC-treated water to obtain the final, purified product (seePurification Protocol Flowchart)

Optional Materials:

-   -   Crude small nucleic acids preparation for purification    -   Materials for generating long dsRNA template    -   Materials for digestion of long dsRNA template to generate crude        siRNA product    -   Chemically synthesized siRNA    -   EtOH (95 or 100%)    -   UltraPure™ Glycogen (20 μg/μl) (Invitrogen cat #10814-010)

Purification Protocol Flowchart

The Small Nucleic Acids Purification System is designed to purifyMicro-RNA molecules such as micro RNA, tiny RNA, small nuclear RNA,guide RNAs, telomerase RNA, small non-mRNA, catalytic RNA, and smallregulatory RNAs (such as aptmaer). Also, RNAi molecules RNaseIII-generated diced siRNA (15-16 bp), Dicer-generated siRNA (21-23 bp),other short hairpin RNA, and small temporary regulatory RNA can bepurified with the Small Nucleic Acids Purification System. Single-ColumnProtocol** Two-Column Protocol* Add 150 μl of Binding Buffer to 50 μlAdd 50 μl of Binding Buffer to 50 μl of of sample reaction volume* andsample reaction volume* and mix it well. mix it well (Total volume: 200μl) (Total volume 100 μl) Add 600 μl of EtOH (95-100%) Add 50 μl of EtOH(95-100%) (Final EtOH concentration 71-75%, (Final EtOH concentration31-33%, total volume: 800 μl) sample volume: 150 μl) Mix sample well andload onto spin column Centrifuge at 20,000 × g for 1 min Expectedrecovery volume: Expected recovery volume: ca. 750 μl ca. 130 μl Removespin column from collection tube Add 185 μl of EtOH (95-100%) to pass-through and mix it well. (Final EtOH conc. ca. 70-74%) Load sample onto2^(nd) column Centrifuge at 20,000 × g for 1 min Wash spin column with500 μl of 1× Wash Buffer Repeat the washing step (optional) Centrifugeat 20,000 × g for 1 min to dry the filter Add 100 μl of Elution Bufferto Add 100 μl of DEPC-treated water to dried spin column and incubate atdried spin column & incubate at ambient ambient temperature for 1 mintemperature for 1 min Centrifuge at 20,000 × g for 1 min Expectedelution volume: ca. 95 μl The eluate contains the purified, short dsRNAEtOH precipitation of short nucleic acids: a. Add 200 μl of ice cold100% EtOH and 1 μl glycogen solution (20 μg/μl). b. Incubate at −20° C.for 15 min and centrifuge for 15 min at 20,000 × g c. Discardsupernatant carefully and wash pellet with 0.5 ml of 70% EtOH d.Centrifuge for 10 min at 20,000 × g e. Discard supernatant and air drypellet Resuspend pellet of purified, short dsRNA in 50 μl (or desirableamount) of DEPC-treated water*Higher sample reaction volumes may require proportionally increasedBinding Buffer and EtOH volumes. (Two-Column protocol provide here isscaled down procedure from siRNA purification kit module ofBlock-iT(Dicer RNAi Kit). Either EtOH or isopropanol can be used# to mixing step with Binding Buffer.**Single column purification will limit its reaction volume to 50 μlreaction. (up to 10 μg of dsRNA reaction).

Please see detail description of Purification of Small NucleicAcids-General Consideration in Results and Discussion section.

Materials and Methods

Generation of dsRNA and siRNA

Crude siRNA needed for purifications was generated in a two-stepprocess. First, in-vitro T7 RNA polymerase transcription reaction wasused to generate the individual strands that form dsRNA, which then, ina second reaction, served as a template for either Dicer or RNase IIIdigestion yielding crude siRNA preparations that were used forpurification with the new kit. The genes of LacZ (Accession number:AY150267) and Luciferase (Accession number: AAL30778.1) were selected asthe target genes for siRNA inhibition. LacZ dsRNA template was generatedas follows: (1) PCR was performed with lacZ gene-specific primer 1(5′-ACC AGA AGC GGT GCC GGA AA-3′ (SEQ ID NO: 49)) and primer 2 (5′-CCACAG GGG ATG GTT CGG AT-3′ (SEQ ID NO: 50)), (2) PCR was performed toincorporate T7 sequences at both ends of the amplicon generated in step1 with Primer 3 (5′-GAC TCG TAA TAC GAC TCA CTA TAG GGA CCA GAA GCG GTGCCG GAA A-3′ (SEQ ID NO: 52)) and primer 4 (5′-GAC TCG TAA TAC GAC TCACTA TAG GGC CAC AGC GGA TGG TTC GGA T-3′ (SEQ ID NO: 53)). The resultingamplicon was purified with Qiagen's PCR clean up kit (QIAquick PCRPurification Kit, cat # 28104) and used as template for the T7 RNApolymerase reverse transcription reaction to generate dsRNA. Long dsRNAwas treated in a final step before Dicer or RNaseIII digestion withDNase I and RNaseA to remove template DNA and unhybridizedsingle-stranded RNA. Luciferase specific dsRNA was generated analogouslyusing the following primer sets: primer 5 (5′-TGA ACA TTT CGC AGC CTACC-3′ (SEQ ID NO: 51)) and primer 6 (5′-GCC ACC TGA TAT CCT TT-3′ (SEQID NO: 54)) for the first round of PCR, primer 7 (5′-GAC TCG TAA TAC GACTCA CTA TAG GGT GAA CAT TTC GCA GCC TAC C-3′ (SEQ ID NO: 55)) and primer8 (5′-GAC TCG TAA TAC GAC TCA CTA TAG GGG CCA CCT GAT ATC CTT T-3′ (SEQID NO: 56)) for the second round of PCR. Plasmids containing the LacZand luciferase gene used as templates (pcDNA 1.2/V5/GW-lacZ andpcDNA5-FRT-luc). These two plasmids were also used for transfection toserve as reporter plasmid for functional testing. The two plasmids usedare components of the BLOCK-iT™ Dicer RNAi Kit (Invitrogen, cat. #K3600-01). Double-stranded RNA, which was to serve as template for siRNAgeneration, was purified using the glass fiber filter columns developedfor siRNA purification as well as with Ambion's purification columns andprotocol. Purified dsRNA template was digested with either Dicer(Invitrogen) or RNase III (Ambion) to generate functional siRNA. Thelatter was purified using the single-column as well as the two-columnprotocol outlined above.

Mammalian Cell Culture and Transfection

For functional testing GripTite™ 293 MSR cells (Invitrogen, cat. #R79507) and F1pIn 293 cells were used. GripTit™ e 293 MSR cells werecultured in DMEM containing 4 mM L-glutamine, 10% FBS, and 600 μg/mlgeneticin (Invitrogen, cat.# 11811-023). In co-transfection experiments100 ng of each reporter plasmid (see above) was co-transfected witheither unpurified siRNA, purified siRNA, or synthetic siRNA specific forlacZ or for Green Fluorescent Protein (GFP) into 90% confluent GripTite™293 cells plated at 2×105 cells/well. F1pIn 293 cells (F1pIn 293 luc)expressing luciferase from a single integrated copy were used to testluciferase specific siRNA. LacZ activity was also monitored as a controlto assess any general, non-specific changes in mRNA expression. F1pIn293 cells were cultured in DMEM containing 4 mM L-glutamine, 10% FBS,and 100 μg/ml hygromycin B (Invitrogen, cat. #10687-010). Cells wereseeded in 24-well plates and grown to 30−50% confluence beforetransfection with siRNA.

β-Galactosidase and Luciferase Assays

Activity and specificity of siRNA transfected was assessed by monitoringthe activity of the reporter gene products luciferase andα-galactosidase. One to two days after transfection the medium wasremoved from each well of the 24-well plates and replaced with 500 μlcold luciferase lysis buffer from Promega (25 mM Tris-HCl pH 8.0, 0.1 mMEDTA pH 8.0, 10% v/v glycerol, 0.1% v/v Triton X-100). Plates were thenfrozen at −80° C. for at least 1 hour. Samples were thawed for 30 min atRT and 50 μl (for luciferase assay) or 10 μl (for β-galactosidase assay)were transferred to a black 96-well plate. For,β-galactosidase, 90 μl ofReaction Dilution Buffer containing 1% (v/v) Galacton-Plus® (AppliedBiosystems, cat # T1006) was added to each sample and incubated for 30min at room-temperature. Luminescence was measured on a MicroLumat Plusluminometer using Winglow v.1.24 software (EG&G Berthold). Forluciferase, either 50 μl of Luciferase Assay Reagent (Promega, cat #E1483) or 100 μl Accelerator II (Tropix) were injected per well andreadings were taken for 5 seconds after a 2-second delay.

Other Materials Used

-   i. Silencer siRNA Cocktail Kit (Cat. no. 1625, Ambion Inc.)-   ii. RNA purification Column 1 and 2 (Cat. no., T510004, T510005,    Gene Therapy Systems, Inc.)-   iii. Yeast tRNA (Cat. no. 15401-011, Invitrogen Inc.)-   iv. E-Gel 4% (Cat. no. G5018-04, Invitrogen Inc.)-   V. 10 bp DNA ladder (Cat. no. 10821-015, Invitrogen Inc.)

Results and Discussion

Purification of Small Nucleic Acids—General Considerations

Commercially available kits for the isolation and purification ofdouble-stranded nucleic acids, RNA as well as DNA, generally do notaddress the need for purification of short double-stranded nucleicacids. A notable exception is the use of size exclusion filtrationtechnology. However, this technology suffers from several drawbacks(limited automation capabilities, broad cut-off size ranges, lowrecoveries, etc.) that have limited its use. Short double-strandednucleic acids shall be defined here as nucleic acids that are shorterthan about 100 bp in length. Ribonucleic acids falling into thiscategory include, but are not limited to, RNA species that are describedin the literature as tiny RNA, small RNA (sRNA), non-coding RNA (ncRNA),micro-RNA (mRNA), small non mRNA (smRNA), functional RNA (fRNA),transfer RNA (tRNA), catalytic RNA such as ribozymes, small nucleolarRNA (snRNA), short hairpin RNA (shRNA), small temporally regulated RNA(strRNA), aptamers, and RNAi molecules including without limitationsmall interfering RNA (siRNA). With recent developments in the field ofRNAi/siRNA technology, a particular need for the purification of siRNAfrom crude enzymatic preparations of siRNA has become apparent. Smallinterfering RNAs (siRNA) are small dsRNA molecules in the size range ofapproximately 12 to 25 bp, which can be generated either enzymaticallyor chemically (Elbashir et. al., 2001). Short deoxyribonucleic acidspotentially requiring purification may comprise, but are not limited to,dsDNA molecules such as adapters, linkers, short restriction fragmentsand PCR products. The purification system described here is based onnucleic acids binding to a glass fiber filter under controlledconditions permitting size-dependant, efficient, high-recovery,high-purity purification of short nucleic acids.

Two general approaches for the purification of small nucleic acids, areoutlined in the purification protocol flowchart above pertaining to thepurification of enzymatically-generated siRNA. Using a single-columnprotocol, all double-stranded nucleic acids exceeding a length ofapproximately 10 base pairs are bound to the glass fiber matrix duringan initial step in the presence of a chaotropic salt, which is containedin the Binding Buffer, and EtOH in excess of 70% (v/v). The binding stepis followed by a wash step, which removes non-nucleic acids componentsfrom the target nucleic acids bound to the glass fiber matrix. In athird step small nucleic acids are selectively eluted in Elution Buffercontaining a controlled amount of EtOH that is specific for the releaseof the targeted nucleic acids size range. Nucleic acids exceeding thetargeted size range will remain bound to the glass fiber filter. In thecase of siRNA, either generated by Dicer or RNaseIII digestion of largerdsRNA template molecules, the optimal concentration of EtOH wasdetermined to be 25% (v/v). Use of the single-column protocol for smallnucleic acids purification typically employs a final EtOH precipitationstep in order to remove chaotropic salts, which are present in theElution Buffer, followed by resuspension of purified nucleic acids in abuffer of choice.

In the two-column protocol double-stranded nucleic acids fragmentsexceeding a length of approximately 30 base pairs are bound to the glassfiber matrix during the initial binding step, while fragments shorterthan approximately 30 base pairs are washed through the glass fibermatrix and are recovered in the flow through. This size fractionation isachieved by applying the mixture of nucleic acids fragment of varioussizes in a Binding Buffer containing a chaotropic salt and a controlledconcentration of EtOH. In the case of siRNA the optimal concentration ofEtOH was determined to be approximately 33% (v/v). The size cut-off forflow through of short nucleic acids can be fine tuned by adjusting therelative amount of EtOH contained in the binding solution. IncreasedEtOH concentrations result in retention of shorter nucleic acidfragments on the glass fiber filter, while decreased EtOH concentrationsin the binding solution result in the elution of larger nucleic acidsfragments. In a second step the EtOH concentration of the flow throughfrom the first column containing the small nucleic acids of interest isincreased to >70% (v/v) and applied to a second glass fiber filtercolumn. Under these conditions small, double-stranded nucleic acids arebound to the matrix of the second filter column. This second bindingstep is followed by a wash step, which removes any remaining non-nucleicacid components from the targeted small nucleic acids bound to the glassfiber matrix. In a final step the targeted small nucleic acids areeluted off the glass fiber matrix at low ionic strength with water.

The single-column and the two-column protocol provide two alternativesfor the purification of small nucleic acids molecules. The single-columnprotocol is more economical as it uses only one filtration step.However, this protocol typically employs a final EtOH precipitationstep, which is more time consuming than a filtration step and holds therisk of incomplete nucleic acid precipitation. On the other hand, EtOHprecipitation is generally considered to yield a cleaner nucleic acidpreparation. The two-column protocol, while more costly per samplepurification, is generally faster than the single-column protocol byvirtue of avoiding the EtOH precipitation step.

Single-Column Protocol: EtOH Fractionation of Crude siRNA

Experimental Setup

Crude lacZ siRNA, which was generated from 1 μg of dsRNA template in a50-μl reaction volume according to the procedure outlined above, wasmixed with 50 μl of Binding Buffer and 100 μl of EtOH at variousconcentrations. Final EtOH concentrations ranged from 5-50%. Sampleswere applied to spin columns, centrifuged, and the flow-through wascollected and analyzed on a 4% E-Gel after EtOH precipitation of nucleicacids in the presence of glycogen.

Results and Discussion

See FIG. 22. Lane 1 in FIG. 22 shows a 10-bp DNA ladder for sizereference. The 20- and 30-bp fragments are marked. The crude lacZ/Dicerreaction is shown in lane 2. Undigested, 1-kb dsRNA template migratesclose to the well. The undigested material generally accounts for asignificant portion of the initial starting material after the dicingreaction, i.e. the Dicer reaction does not completely digest dsRNAsubstrate even after prolonged reaction times. Since the presence ofundigested and partially digested dsRNA substrate is incompatible withcell viability, purification is essential. In the case shown, undigestedtemplate accounts for more than 50% of the starting material. The dsRNADicer reaction product, which has a length of 21-23 bp, migrates betweenthe 20- and 30-bp fragments of the DNA ladder shown in lane 1. Reactionintermediates, partially digested dsRNA template which are apparent as abackground smear in the lane, migrate between the undigested templateand the siRNA reaction product. At an EtOH concentration of 5% in theBinding Buffer most of the 1-kb dsRNA template as well as shorter dsRNAmolecules do not bind to the filter matrix and are consequentlyrecovered in the flow-through (FIG. 22, lane 3). Increasing ethanolconcentration in the Binding Buffer leads to the binding ofprogressively shorter dsRNA fragments to the filter matrix resulting inthe selective binding of unwanted longer dsRNA fragments and selectiveflow-through of targeted siRNA molecules (FIG. 22, lanes 4-12). At anEtOH concentration exceeding 20% it appears that only targeted 21-23 bpsiRNA selectively elute while longer dsRNA fragments are retained on thefilter. At EtOH concentrations of 20-30% (lanes 6-8) recovery appears tobe efficient, while at higher ethanol concentrations (35-50%, lanes9-12) recovery decreases due to binding of even short dsRNA molecules atthese elevated EtOH concentrations. At EtOH concentrations exceeding 50%siRNA showed increasing affinity for the glass fiber matrix of thefilter. Efficient binding of siRNA can be achieved with EtOHconcentrations of 70% or more even for the shorter siRNA productsderived from RNase III digestion (see below).

FIG. 22 shows fractionation of double-stranded RNA using differentethanol concentrations. Flow-through samples were analyzed on a 4% E-Gelafter EtOH precipitation in the presence of glycogen and resuspension inRNase-free water.

-   -   Lane 1: 10 bp DNA Ladder (Invitrogen Cat # 18021-015)    -   Lane 2: Crude lacZ/Dicer siRNA reaction with 1-kb dsRNA template    -   Lane 3: Flow-through of 5% EtOH-containing Binding Buffer    -   Lane 4: Flow-through of 10% EtOH-containing Binding Buffer    -   Lane 5: Flow-through of 15% EtOH-containing Binding Buffer    -   Lane 6: Flow-through of 20% EtOH-containing Binding Buffer    -   Lane 7: Flow-through of 25% EtOH-containing Binding Buffer    -   Lane 8: Flow-through of 30% EtOH-containing Binding Buffer    -   Lane 9: Flow-through of 35% EtOH-containing Binding Buffer    -   Lane 10: Flow-through of 40% EtOH-containing Binding Buffer    -   Lane 11: Flow-through of 45% EtOH-containing Binding Buffer    -   Lane 12: Flow-through of 50% EtOH-containing Binding Buffer

CONCLUSION

Removal of undigested and partially digested dsRNA substrate andhigh-purity recovery of Dicer-generated siRNA can be achieved bycontrolling EtOH concentration in the final binding solution. Optimalresults are achieved with final EtOH concentrations ranging from 20-30%in the binding solution.

Functional Testing of Purified siRNA (Single-Column and Two-Column

Protocol Comparison)

Experimental Setup

As outlined above in the Purification Protocol Flowchart, twoalternative purification approaches are feasible depending mainly onindividual preferences regarding ethanol precipitation and proceduretime. In the following experiments, which are illustrated in FIGS.19A-19C, lacZ siRNA was purified according to the single- and column andtwo-column protocols described earlier. The siRNA obtained was testedfor specificity and functionality in transfection experiments usingGripTite™ 293 MSR cells, which contained either luciferase orβ-galactosidase gene constructs in reporter plasmids, as described indetail above. In the case of the single-column purification protocol,elution was performed with Elution Buffer containing ethanol at finalconcentrations of between 5 and 30%. Transfection experiments wereperformed in duplicate.

Results and Discussion

FIG. 23A shows gel analysis results of crude lacZ siRNA, siRNA purifiedusing the two-column protocol, various fractions of the single-columnpurification protocol, as well as chemically synthesized siRNA analyzedon a 4% E-Gel, which were used for functional testing. Green FluorescentProtein (GFP) siRNA, by virtue of being chemically synthesized, does notcontain any long dsRNA impurities. The siRNA that was purified with thetwo-column protocol and siRNA fractions eluted with 20, 25, and 30% EtOHusing the single-column protocol appear to be devoid of intermediateDicer reaction products and full-length dsRNA template. Therefore, thesesiRNA preparations are expected to be potent and specific in thesuppression of their target genes and are not expected to exhibit any ofthe adverse effects associated with the presence of long dsRNA.Unpurified lacZ siRNA contains significant amounts of undigested andpartially digested long dsRNA molecules and is hence expected to resultin cell death upon transfection. Likewise, siRNA purified with thesingle-column protocol and eluted with Elution Buffer containing 5, 15,and 20% EtOH is expected to result in cell death, albeit at decreasingdegrees as ethanol concentration increases.

FIG. 23 A:

-   -   Lane 1: 10 bp DNA Ladder (Invitrogen Cat # 18021-015)—The 10-bp        fragment only shows as a faint band.    -   Lane 2: Chemically synthesized, unpurified Green Fluorescent        Protein (GFP) siRNA    -   Lane 3: Crude lacZ siRNA reaction mixture    -   Lane 4: LacZ siRNA purified using the two-column protocol (see        flowchart above)    -   Lane 5: LacZ siRNA eluted with 5% EtOH-containing Elution Buffer        according to the single-column protocol    -   Lane 6: LacZ siRNA eluted with 10% EtOH-containing Elution        Buffer according to the single-column protocol    -   Lane 7: LacZ siRNA eluted with 15% EtOH-containing Elution        Buffer according to the single-column protocol    -   Lane 8: LacZ siRNA eluted with 20% EtOH-containing Elution        Buffer according to the single-column protocol    -   Lane 9: LacZ siRNA eluted with 25% EtOH-containing Elution        Buffer according to the single-column protocol    -   Lane 10: LacZ siRNA eluted with 30% EtOH-containing Elution        Buffer according to the single-column protocol

The effects of lacZ siRNA on luciferase activity are shown in FIG. 23B.This is a negative control experiment. Since lacZ siRNA does not havesequence homology to the luciferase gene, its activity should remainunperturbed by the presence of lacZ siRNA. Any changes in luciferaseactivity will thus be attributed to nonspecific effects such as theeffect that the presence of long dsRNA may have on the transfected cellsor the effects of transfection itself. Cells that do not carry thereporter plasmid for luciferase (untransfected) do not exhibitluciferase activity. Cells transfected with the reporter plasmid forluciferase (reporters alone) exhibit baseline luciferase activityserving as a point of reference for the action of siRNA in the followingexperiments. Transfection of cells carrying luciferase reporter plasmidwith chemically synthesized, unrelated GFP siRNA (GFP siRNA) did notalter luciferase activity, as expected. Unpurified, crude lacZ Dicerreaction containing undigested and partially digested long dsRNAresulted in cell death and a concomitant lack of luciferase activity(lacZ dicing reaction). Transfection of target cells with lacZ siRNApurified using the two-column purification protocol (lacZ d-siRNA) didnot suppress luciferase activity as expected. However, nonspecificinduction of luciferase activity by about 40% was apparent. LacZ siRNAobtained by elution with Elution Buffer containing 5, 10, or 15% ethanolusing the single-column protocol (lacZ fract 5, lacZ fract 10, lacZfract 15) resulted in suppression of luciferase activity. Thisobservation, however, is attributed to residual long dsRNA template andpartial digestion products thereof in these fractions eliciting celldeath as observed for unpurified Dicer reactions. The observedsuppression of luciferase activity in these cases is not the result ofspecific siRNA action. LacZ siRNA obtained by elution with ElutionBuffer containing 20, 25, and 30% EtOH did not alter luciferaseactivity. Hence, these fractions of purified siRNA do not elicitnonspecific effects such as induction or suppression of luciferaseactivity upon transfection.

The effects of lacZ siRNA on its target transcripts, as evidenced andmeasurable through the activity of β-galactosidase, are shown in FIG.23C. Cells that do not carry the reporter plasmid for β-galactosidase(untransfected) do not exhibit β-galactosidase activity. Cellstransfected with the reporter plasmid for P-galactosidase (reportersalone) exhibit baseline β-galactosidase activity serving as a point ofreference for the action of siRNA in the following experiments.Transfection of cells carrying β-galactosidase reporter plasmid withchemically synthesized, unrelated GFP siRNA (GFP siRNA) did not alterβ-galactosidase activity. Unpurified, crude lacZ Dicer reactioncontaining undigested and partially digested long dsRNA resulted in celldeath and a concomitant lack of β-galactosidase activity (lacZ dicingreaction). Transfection of target cells with lacZ siRNA purified usingthe two-column purification protocol (lacZ d-siRNA) suppressedβ-galactosidase activity by approximately 70%. LacZ siRNA obtained byelution with Elution Buffer containing 5, 10, or 15% ethanol using thesingle-column protocol (lacZ fract 5, lacZ fract 10, lacZ fract 15)resulted in suppression of β-galactosidase activity. This observation,however, may be attributed to residual long dsRNA template and partialdigestion products thereof in these fractions, eliciting cell death asobserved for unpurified Dicer reactions. In addition, β-galactosidaseactivity in surviving cells may further be suppressed by the presence ofsiRNA specific for the lacZ gene. Thus, while suppression appears to beefficient, it is mainly caused by cell death and not by the specificaction of the siRNA used. LacZ siRNA obtained by elution with ElutionBuffer containing 20, 25, and 30% EtOH did profoundly suppress theactivity of the β-galactosidase enzyme by approximately 80%. In thelatter case cells appeared healthy after transfection with purifiedsiRNA. Hence, these fractions of purified siRNA are highly effective andspecific in the suppression of their targeted mRNA.

Fractionated siRNA samples used were obtained using either thesingle-column or two-column protocol as a means of purification. Theeffects of lacZ siRNA are specific for the β-galactosidase gene due tosequence homologies and a reduction of β-galactosidase activity isexpected as a result of the presence of lacZ siRNA.

FIGS. 23 B and C:

-   -   Untransfected: Cells have not been transfected with reporter        plasmids carrying the luciferase or β-galactosidase gene    -   Reporters alone: Cells have been transfected with reporter        plasmid only, but not with siRNA    -   GFP siRNA: Transfection with chemically synthesized, crude siRNA        specific for the green fluorescent protein gene    -   LacZ dicing reaction: Transfection with crude, unpurified lacZ        siRNA from Dicer reaction    -   LacZ d-siRNA: Transfection with lacZ siRNA purified using the        two-column protocol    -   LacZ frac 5: Transfection with lacZ siRNA from 5% EtOH        containing fraction (single-column protocol)    -   LacZ frac 10: Transfection with lacZ siRNA from 10% EtOH        containing fraction (single-column protocol)    -   LacZ frac 15: Transfection with lacZ siRNA from 15% EtOH        containing fraction (single-column protocol)    -   LacZ frac 20: Transfection with lacZ siRNA from 20% EtOH        containing fraction (single-column protocol)    -   LacZ frac 25: Transfection with lacZ siRNA from 25% EtOH        containing fraction (single-column protocol)    -   LacZ frac 30: Transfection with lacZ siRNA from 30% EtOH        containing fraction (single-column protocol)

Purification of siRNA Generated by Dicer or RNase III

Experimental Setup

One-kb dsRNA transcript of either lacZ or luciferase was incubated withDicer or RNaseIII to generate double-stranded siRNA products. Dicerreactions were carried out using a protocol as described in theBLOCK-iT™ Complete Dicer RNAi Kit (Invitrogen cat. # K3650-01).Digestion with RNaseIII (Ambion, cat. # 2290) was performed according tothe manufacturer's suggestions. Crude RNase III and Dicer siRNAreactions were purified using the single-column and two-columnpurification protocol and subsequently tested for functionality.

Results and Discussion

The Dicer enzyme is a member of the RNaseIII family of ribonucleases anddigests long dsRNA templates into 21-23 nucleotide, double-strandedsiRNA that have been shown to function as key intermediates intriggering sequence specific RNA degradation during posttranscriptionalgene silencing. Likewise, RNaseIII digests long dsRNA templates intoshort double-stranded siRNA molecules. However, the siRNA generated byRNaseIII is generally only approximately 12-15 base pairs long. Dicerenzyme is found in all eukaryotic cells and RNaseIII is mainly found inprokaryotes. Dicer enzyme is speculated to bind to the ends of longdsRNA and progressively cleave the template dsRNA. The mode of action ofRNaseIII may involve random cleavage of template dsRNA into smaller,compared to the Dicer enzyme, 12-15 bp siRNA fragments. The RnaseIIIenzyme is considerably more active than the corresponding Dicer enzyme,which leads to complete digestion of template dsRNA by RNaseIII enzyme,while Dicer enzyme, even after prolonged digestion, results in onlyincomplete digestion of template dsRNA. These findings are illustratedin FIG. 24A. Neither enzyme requires ATP for function. However, bothenzymes require divalent metal cations and a specific, optimal pH rangefor optimal activity, which are provided by enzyme-specific reactionbuffers. The purification procedures for siRNA used here result in theremoval of proteins and buffer components. The purified siRNA isresuspended in RNase-free water in the final purification stepindependent of the purification protocol used. Short interfering RNAgenerated by the action of RNase III as well as by Dicer enzyme weresuccessfully purified using Invitrogen spin columns applying either thesingle- or two-column purification protocol as shown in FIG. 24B.

FIGS. 24A and 24B show purification of siRNA generated with Dicer andRNaseIII

FIG. 24A:

-   -   Lane 2: unpurified lacZ siRNA cleaved by RNaseIII,    -   Lane 3 unpurified luciferase siRNA cleaved by RNaseIII,    -   Lane 4: unpurified lacZ siRNA cleaved by Dicer,    -   Lane 5: unpurified luciferase siRNA cleaved by Dicer

FIG. 24B:

-   -   Lane 1, 5,9: lacZ siRNA cleaved by RNaseIII,    -   Lane 2, 6,10: luciferase siRNA cleaved by RNaseIII,    -   Lane 3, 7,11 lacZ siRNA cleaved by Dicer,    -   Lane 4, 8,12 luciferase siRNA cleaved by Dicer

CONCLUSION

Short interfering RNA generated by digestion of long dsRNA templateswith either Dicer or RNaseIII enzyme can be efficiently purified usingthe single-column or two-column purification protocol.

Functional Testing of SiRNA Preparations with F1pIn 293 luc Cells

Experimental Setup

Short interfering RNA was generated by digestion of long dsRNA templates(1 μg) with either RNase III or Dicer enzyme. Samples were purifiedusing the single-and two-column purification protocol. Concentrations ofpurified siRNA were determined by A260 measurements and 20 ng ofpurified sample was used for each transfection into F1pIn 293 luc cells.

Results and Discussion

Transfection experiments were performed in 5 experimental groups witheach experiment being conducted in duplicate. Experimental group 1consisted of two control reactions of mock transfected (Mock), i.e.transfection with transfection agent but without siRNA, as well as F1pIn293 luc cells expressing baseline levels of luciferase (Untransfected).The luciferase activity was measured in these latter two experiments andserved as reference for luciferase activity that was determined in group2-5 experiments. In experimental groups 2 and 3 the effect of luciferaseactivities by Dicer generated luciferase specific siRNA (luc siRNA) andβ-galactosidase specific siRNA (lacZ siRNA) were assessed. Likewise, ingroups 4 and 5 the effect of siRNA generated with RNaseIII enzyme onluciferase activity was assessed.

siRNA (20 ng luc Dicer reaction) resulted in cell death and nonspecificlack of luciferase activity (see FIG. 25). SiRNA that was purified usingthe single-column purification protocol, where siRNA was eluted withElution Buffer containing 25 or 30% EtOH (20 ng luc Dicer 25% pur. & 20ng luc Dicer 30% pur.), resulted in efficient suppression of luciferaseactivity by more than 80%. Equally efficient suppression was achievedwith siRNA purified using the two-column purification protocol (20 ngluc Dicer 2 col. pur.) and with the latter siRNA that was subjected toan additional step of EtOH precipitation (20 ng luc d-siRNA (EtOH)).Thus, luciferase specific siRNA purified with either the single-columnor two-column purification protocol is highly potent in suppressing theactivity of luciferase.

As shown in experimental group 3 in FIG. 25, all purified andβgalactosidase-specific siRNA samples generated with the Dicer enzymefailed, as expected, to significantly change expression levels of theluciferase gene as determined by assessing luciferase activity.Variations in the activity of the luciferase enzyme caused byβ-galactosidase-specific siRNA were generally less than 10%. As observedearlier, unpurified Dicer-generated siRNA (20 ng luc Dicer reaction)resulted in cell death and nonspecific lack of luciferase activity.Thus, P-galactosidase-specific siRNA purified with the single-column ortwo-column protocol does not cause any significant nonspecific inductionor suppression of bystander proteins, in this case luciferase.

In experimental groups 4 and 5 the effect of luciferase specific siRNA(luc siRNA) as well as β-galactosidase enzyme specific siRNA (lacZsiRNA) generated with RNaseIII enzyme (Ambion) on luciferase activitywas assessed. Unlike unpurified Dicer reactions, which containsignificant amounts of undigested or partially digested long dsRNAtemplate, unpurified RNaseIII reactions do not contain significantamounts of undigested or partially digested long dsRNA template aspreviously shown in FIG. 24A. Consequently, transfection with unpurifiedRNaseIII reaction products (20 ng luc RNaseIII reaction & 20 ng lacZRNaseIII reaction) does not lead to cell death and concomitantnonspecific reduction of luciferase activity. RNase III digestionproducts are in the 13-15 bp size range, which is well below the sizerange reported for potent siRNA (˜20-23 bp). Consequently, purifiedRNaseIII-generated luciferase specific siRNA (20 ng luc RNaseIII 25%pur., 20 ng luc RNaseIII 30% pur, and 20 ng luc RNaseIII 2 col. pur.)suppressed luciferase activity by only approximately 25% under theconditions used here. This lack of efficient suppression at the siRNAconcentrations used may be attributable to a lack of functional siRNApresent after digestion with RNaseIII since the siRNA size generated byRNaseIII is less than 20 base pairs (see FIGS. 24A and 24B). Czaudernaet al.(Nucleic Acids Research 2003) reported that synthetic siRNAsshorter than 19 base pairs in size were not effective in suppressinggene expression. Concentrations of up to 200 ng/transfection ofRNaseIII-generated siRNA were tested. However, even at these elevatedamounts no significant suppression was observed. As shown inexperimental group 5, neither unpurified nor purified lacZ siRNA thatwas generated by digestion of long dsRNA templates with RNaseIII had anyeffect on luciferase activity.

Functional Testing of siRNA Preparations with GripTite™ MSR Cells

Experimental setup

Two reporter plasmids (see above) expressing luciferase andβ-galactosidase, respectively, were co-transfected into GripTite™ 293MSR cells with siRNA specific for luciferase mRNA (luc) orβ-galactosidase mRNA (lacZ) generated by Dicer or RNaseIII using theexperimental scheme described in the previous experiment. Luciferase andβ-galactosidase activity was determined as described above to assess theeffect of specific siRNA preparations on the expression of the two genetranscripts under investigation.

Results and Discussion

Results presented in FIG. 26A demonstrate the effect of different lucsiRNA and lacZ siRNA preparations generated with Dicer and RNaseIIIenzyme on β-galactosidase activity. The results obtained are in goodagreement with the results shown in FIG. 25. In brief, GripTite 293 MSRcells transfected with the reporter plasmid alone (Reporters Only)exhibited reference levels of β-galactosidase activity. Cells nottransfected with the reporter plasmid (Mock) did not yield anyβ-galactosidase activity. As seen previously, crude Dicer reactions (20ng luc Dicer reaction & 20 ng lacZ Dicer reaction) caused cell death andnonspecific suppression of β-galactosidase activity, while this effectwas not observed with crude RNaseIII reactions (20 ng luc RNaseIIIreaction & 20 ng lacZ RNaseIII reaction). All preparations of purified,Dicer-generated lacZ siRNA efficiently suppressed expression ofα-galactosidase activity by more than 80%. On the other hand, neitherpreparation of the negative control luc siRNA affected β-galactosidaseactivity to any significant degree. SiRNA generated by digestion withRNaseIII elicited similar responses to those observed above. Suppressionof β-galactosidase activity by lacZ siRNA preparations was inefficientwith maximum suppressions of 40%. However, from the results shown inFIG. 26A it is apparent that luciferase specific siRNA preparationsgenerated with RNaseII enzyme caused significant nonspecific inductionof the β-galactosidase gene.

Results presented in FIG. 26B demonstrate the effect of different lucsiRNA and lacZ siRNA preparations generated with Dicer and RNaselllenzyme on luciferase activity. The results obtained are in goodagreement with the results shown in FIG. 25. In brief, GripTite 293 MSRcells transfected with the reporter plasmid alone (Reporters Only)exhibited reference levels of luciferase activity. Cells not transfectedwith the reporter plasmid (Mock) did not yield any luciferase activity.Crude Dicer reactions (20 ng luc Dicer reaction & 20 ng lacZ Dicerreaction) caused cell death and nonspecific suppression of luciferaseactivity, while this effect was not observed with crude RNaseIIIreactions (20 ng luc RNaseIII reaction & 20 ng lacZ RNaseIII reaction).All preparations of purified, Dicer-generated luc siRNA efficientlysuppressed expression of luciferase activity by more than 90%. On theother hand, neither preparation of the negative control lacZ siRNAsuppressed luciferase activity. However, in the series of experimentsshown here, luciferase activity was stimulated by up to 40% byβ-galactosidase specific lacZ siRNA. SiRNA generated by digestion withRNaseIII elicited similar responses to those observed above. Thesuppression of luciferase activity by luc siRNA preparations wasinefficient with maximum suppressions of approximately 20%. The effectsof lacZ siRNA preparations generated with RNaseIII on luciferaseactivity were inconsistent with both induction (20 ng lacZ RNaseIII 25%pur.) and suppression (20 ng lacZ RNaseIII 30% pur. & 20 ng lacZRNaseIII 2 col. pur.) being observed.

CONCLUSION

SiRNA generated by digestion of long dsRNA templates with Dicer enzymeand purified using either the single-column or two-column purificationprotocol efficiently suppressed gene specific expression with minimalnonspecific induction of bystander proteins. Short interfering RNAgenerated by digestion of long dsRNA templates with RNaseIII enzyme,while efficiently purified with either the single-column or two-columnpurification protocol, did not perform well under the experimentalconditions used here.

Column Capacity and Recovery Determination

Experimental setup.

These experiments were intended to determine the recovery of RNA, tRNAand a 1-kb dsRNA fragment, after binding to the glass fiber matrix ofthe spin column as a function of elution volume. The experiments werealso designed to provide information about the general loading capacityof the spin column for short double-stranded nucleic acids and longdsRNA fragments, the latter are used as templates for RNase digestionassays. In order to assess the column capacity for siRNA, yeast tRNA wasused, because it was available in the quantities needed. Yeast tRNAconstitutes a sensible alternative for column testing to siRNA as itslinear, single-stranded size is approximately 75 nucleotides that areinvolved in extensive secondary structure formation, i.e. tRNA ispresent predominantly in dsRNA form. The tRNA used here migrates like a40-bp double-stranded nucleic acid fragment on agarose gels. Theefficiency of recovery and approximate loading capacity was alsodetermined for a 1-kb dsRNA fragment. These long dsRNA fragments serveas templates for Dicer and RNaseIII digestion and require purificationafter clean up of the transcription reaction with DNaseI and RNaseA andprior to Dicer/RNaseIII digestion for generating siRNA. Purificationswere carried out using the single-column purification protocol with10-1000 μg of yeast tRNA or 4-240 μg of the 1-kb dsRNA fragment. BounddsRNA was eluted from the spin columns with either a single elution of100 μl DEPC-treated water or two successive elutions of 50 μlDEPC-treated water. Amounts of eluted RNA were quantified by A260measurements and compared to the initial amount of RNA loaded.

Results and Discussion

Ten μg of tRNA were eluted with either a single 100-μl elution or two50-μl elutions with an efficiency exceeding 90% (FIG. 27A). Amounts oftRNA of up to 1 mg can be eluted with efficiencies of approximately 80%,independent of whether a single 100-μl elution or two 50-μl elutionswere used. Recovery can be further increased to about 95% with a second100-μl elution or a third 50-μl elution. It shall be noted that foryeast tRNA amounts in excess of about 100 μg the addition of BindingBuffer and EtOH to the sample results in precipitation of presumablytRNA. The results shown in FIG. 27A demonstrate that tRNA, and bycorrelation siRNA, can be recovered almost quantitatively from the spincolumn matrix by elution with DEPC-treated water. Also, the results showthat the column capacity exceeds 1 mg for tRNA/siRNA. FIG. 27B shows therecovery results obtained with a 1-kb dsRNA fragment loaded at amountsranging from 4-240 μg. Independent of whether a single 100-μl elution ortwo 50-μl elutions were used recovery efficiency was about 90%. It shallbe noted that the long dsRNA fragment was more susceptible to form aprecipitate after the addition of Binding Buffer and EtOH than tRNA.Loading of dsRNA amounts exceeding about 500 μg resulted inprogressively decreasing recoveries with either a single 100-μl elutionor two 50-μl elutions, which could be improved with additional

CONCLUSION

Short dsRNA, e.g., siRNA or tRNA, as well as long dsRNA fragments can beefficiently eluted after binding to the spin column with DEPC-treatedwater. No major differences in recovery were observed for either asingle elution with 100 μl or two successive 50-μl elutions.

Clean-up of Long dsRNA Substrate and tRNA

Experimental Setup

Three different sizes of long dsRNA (100, 500 and 1 kb) of the lacZ genewere generated by T7 polymerase reactions as described above. The 100-bpand 500-bp lacZ dsNRA fragments were generated using primer 1 (seeabove) and primer 9 (5′-GCA TCG TAA CCG TGC ATC 3′ (SEQ ID NO: 57) andprimer 10 (5′ GCG AGT GCC AAC ATG G 3′ (SEQ ID NO: 58), respectively,for the first round PCR. Primer 3 (see above) in combination with primer11 (5′-GAC TCG TAA TAC GAC TCA CTA TAG GTA CTG CAT CG T AAC CGT GCATC-3′ (SEQ ID NO: 59)) and primer 12 (5′-GAC TCG TAA TAC GAC TCA CTA TAGGTA CTG CGA GTG GCA ACA TGG-3′ (SEQ ID NO: 60)), respectively, were usedfor the second round of PCR. The 1-kb dsRNA fragment was generated asdescribed above. All dsRNA fragments generated were cleaned up by DNase1 and RNase A digestion to remove DNA and single-stranded RNA from thereactions before purification using a modified single-column protocol(see below).

Results and Discussion

Long dsRNA intended for Dicer or RNaseIII digestion has to be cleaned upwith DNase I and RNase A to remove DNA and unhybridized single-strandedRNA. Subsequently, the latter enzymes, their digestion products, andbuffer components need to be removed prior to digestion of the longdsRNA templates with Dicer or RNaseIII. A modified version of thesingle-column protocol was used to purify long dsRNA suitable for Dicerand RNaseIII digestion. Binding capacity and recovery of dsRNA from theglass fiber filters was determined previously. Purification results oflong dsRNA and tRNA are shown in FIGS. 28A and 28B.

Purification of dsRNA (50 ul sample)

-   1. Add 150 μl of Binding Buffer and mix well-   2. Add 600 μl of 100% EtOH (Final EtOH concentration of 75%)-   3. Mix well and load onto column-   4. Centrifuge at 14000 rpm for 1 min-   5. Wash with 500 μl of diluted Wash Buffer-   6. Repeat the washing step-   7. Centrifuge at 14000 rpm for 1 min to dry column-   8. Add 100 μl of DEPC-treated water-   9. Wait for 1 min-   10. Centrifuge at 14000 rpm for 1 min to recover dsRNA

FIG. 28A:

-   -   Lane 1: 1 kb Plus DNA Ladder (Invitrogen)    -   Lane 3: 100-bp lacZ dsRNA fragment    -   Lane 5: 500-bp lacZ dsRNA fragment    -   Lane 7: 1-kb lacZ dsRNA fragment

FIG. 28 B:

-   -   Lane 1: 10 bp DNA Ladder (Invitrogen)    -   Lane 3: Unpurified yeast tRNA (0.3 μg)    -   Lane 4: Cleaned-up yeast tRNA (0.3 μg))    -   Lane 6: Unpurified (1.5 μg)    -   Lane 7: Cleaned-up yeast tRNA (1.5 μg))

REFERENCES

-   Kaufman, R. J., Proc Natl. Acad. Sci. USA 96:11693-11695 (1999).-   Denli, A. M. and Hannon, G. J., Trends Biochem. Sci. 28:196-201    (2003).-   Carrington,J. C. and Ambros, V., Science. 301:336-338 (2003).-   Sledz, C. A, et al., Nat Cell Biol. 5:834-839 (2003).-   Illangasekare, M. and Yarus, M., RNA. 5:1482-1489 (1999).-   Elbashir, S. M., et al., Nature 411:494-498 (2001).-   Czauderna, F., et al., Nucleic Acids Res. 31:2705-2716 (2003).-   Elbashir, S. M., et al., EMBO J. 20:6877-6888 (2001).

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for the in vitro generation of RNA molecules, the methodcomprising: (a) generating a reaction mixture in which a firstdouble-stranded DNA molecule is contacted with a second double-strandedDNA molecule under conditions which allow for both strands of one end ofthe first double-stranded DNA molecule to become covalently linked toboth strands of one end of the second double-stranded DNA molecule, (b)incubating the reaction mixture of (a) for a sufficient period of timeto allow for the covalent linking of the first double-stranded DNAmolecule to the second double-stranded DNA molecule, and (c) generatingan RNA transcript from the product of (b) by in vitro transcription,wherein the first double-stranded DNA molecule has promoter activity andis operably connected to the second double-stranded DNA molecule in (b),wherein the first double-stranded DNA molecule and the seconddouble-stranded DNA molecule are covalently linked to each other by atopoisomerase, and wherein the double-stranded DNA molecule produced in(b) does not contain a nick in either strand at the position where thefirst double-stranded DNA molecule and the second double-stranded DNAmolecule are joined.
 2. The method of claim 1, wherein both strands ofone end of the double-stranded DNA molecule product of (b) arecovalently linked by a topoisomerase to both strands of one end a thirddouble-stranded DNA molecule.
 3. The method of claim 2, wherein thedouble-stranded DNA molecules are covalently linked in the order of thefirst double-stranded DNA molecule, the second double-stranded DNAmolecule, and the third double-stranded DNA molecule.
 4. The method ofclaim 2, wherein the third double-stranded DNA molecule encodes apolyadenylation signal.
 5. The method of claim 1, wherein the seconddouble-stranded DNA molecule encodes a polypeptide.
 6. The method ofclaim 1, wherein the second double-stranded DNA molecule is generated bypolymerase chain reaction.
 7. The method of claim 1, wherein one strandof each end of the first double-stranded DNA molecule and the seconddouble-stranded DNA molecule which are joined are topoisomerase-charged.8. The method of claim 1, wherein the topoisomerase is a type IBtopoisomerase or a catalytic domain of a type IB topoisomerase.
 9. Themethod of claim 1, wherein the transcription product of (c) istranslated in vitro to generate a polypeptide.
 10. The method of claim1, wherein the first double-stranded DNA molecule comprises a T7promoter.
 11. The method of claim 1, wherein the first double-strandedDNA molecule comprises a T3 promoter.
 12. A method for the in vitrogeneration of double-stranded RNA molecules, the method comprising: (a)generating a reaction mixture in which a first double-stranded DNAmolecule is contacted with a second double-stranded DNA molecule underconditions which allow for both strands of one end of the firstdouble-stranded DNA molecule to become covalently linked to both strandsof a first end of the second double-stranded DNA molecule, (b)incubating the reaction mixture of (a) for a sufficient period of timeto allow for the covalent linking of the first double-stranded DNAmolecule to the first end of the second double-stranded DNA molecule,and (c) generating a reaction mixture in which a first double-strandedDNA molecule is contacted with a second double-stranded DNA moleculeunder conditions which allow for both strands of one end of the firstdouble-stranded DNA molecule to become covalently linked to both strandsof a second end of the second double-stranded DNA molecule, (d)incubating the reaction mixture of (c) for a sufficient period of timeto allow for the covalent linking of the first double-stranded DNAmolecule to the second end of the second double-stranded DNA molecule,(e) mixing the products of (b) and (d), (f) generating RNA transcriptsfrom the products of (e) by in vitro transcription, and (g) incubatingthe RNA transcripts produced in (f) under conditions which allow for theformation of double-stranded RNA molecules, wherein each of the firstdouble-stranded DNA molecules in (b) and (d) has promoter activity andis operably connected to each of the second double-stranded DNAmolecules in (b) and (d), wherein the first double-stranded DNA moleculeand the second double-stranded DNA molecule are covalently linked toeach other by a topoisomerase, and wherein the double-stranded DNAmolecule produced in (b) does not contain a nick in either strand at theposition where the first double-stranded DNA molecule and the seconddouble-stranded DNA molecule are joined.
 13. The method of claim 12,wherein the RNA transcripts produced in (f) are sense and antisense RNAmolecules.
 14. The method of claim 12, wherein the seconddouble-stranded DNA molecule encodes a polypeptide.
 15. The method ofclaim 12, wherein the second double-stranded DNA molecule is generatedby polymerase chain reaction.
 16. The method of claim 12, wherein onestrand of each of the ends of the first double-stranded DNA molecule andthe second double-stranded DNA molecule which are joined aretopoisomerase-charged.
 17. The method of claim 12, wherein thetopoisomerase is a type IB topoisomerase or a catalytic domain of a typeIB topoisomerase.
 18. The method of claim 12, wherein the firstdouble-stranded DNA molecule comprises a T7 promoter.
 19. The method ofclaim 12, wherein the first double-stranded DNA molecule comprises a T3promoter.
 20. A reaction mixture comprising: (a) a first double-strandedDNA molecule which comprises a promoter, and (b) a seconddouble-stranded DNA molecule, wherein one strand of one end of the firstdouble-stranded DNA molecule is topoisomerase-charged, wherein onestrand of one end of the second double-stranded DNA molecule istopoisomerase-charged, and wherein the topoisomerase-charged ends of thefirst double-stranded DNA molecule and the second double-stranded DNAmolecule are capable of hybridizing to each other.
 21. The reactionmixture of claim 20, wherein the first double-stranded DNA moleculecomprises a T7 promoter.
 22. The reaction mixture of claim 20, whereinthe first double-stranded DNA molecule comprises a T3 promoter.
 23. Thereaction mixture of claim 20, wherein the topoisomerase is a type IBtopoisomerase or a catalytic domain of a type IB topoisomerase.